Postpartum-derived cells for use in treatment of disease of the heart and circulatory system

ABSTRACT

Cells derived from postpartum tissue are disclosed along with methods for their therapeutic use in diseases of the heart or circulatory system are disclosed. Cells may be used therapeuticall in either differentiated or undifferentiated forms, in homogenous cultures, or as populations with other cells, and in conjunction with other bioactive factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit of U.S. Provisional Application Ser. No. 60/483,264,filed Jun. 27, 2003, the entire contents of which are incorporated byreference herein. Other related applications include the followingcommonly-owned, co-pending applications, the entire contents of each ofwhich are incorporated by reference herein: U.S. Application No.[ETH-5073 NP1], filed Jun. 25, 2004; U.S. Application No. [ETH-5073NP2], filed Jun. 25, 2004; U.S. Application No. [ETH-5073 NP3], filedJun. 25, 2004; U.S. Application No. [ETH-5073 NP4], filed Jun. 25, 2004;U.S. Application No. [ETH-5073 NP5], filed Jun. 25, 2004; U.S.Application No. [ETH-5073 NP8], filed Jun. 25, 2004; and U.S.Provisional Application No. 60/555,908, filed Mar. 24, 2004.

FIELD OF THE INVENTION

The invention relates to postpartum-derived cells that have thepotential to divide, and to differentiate along mesenchymal lineage,towards cardiomyogenic, angiogenic and vasculogenic phenotypes. Theinvention also relates to methods for the use of such postpartum-derivedcells in therapeutic treatment of diseases of the heart and circulatorysystem.

BACKGROUND OF THE INVENTION

It is generally recognized that the cells which comprise the heartmuscle in mammals are post-mitotic. This leads to difficulties ininjured or diseased heart muscles, which are largely unable to repairdamaged cells that become necrotic. After such damage, the efficiencyand output of the heart muscle are decreased, placing additional stresson the heart, leading to further damage and necrosis, and ultimately toheart failure.

The downward spiral from healthy heart to failing heart can result froma number of conditions including physical injury, heart disease,including congenital heart disease, and infections of the heart orcirculatory tissue. Diseases of the heart and circulatory system areoften ultimately fatal, particularly conditions which result in heartfailure, for example cardiomyopathies. At present there is no cure formost such conditions and many patients require, for example, ventricularassist devices and eventually heart transplants.

Presently, there is interest in using either stem cells, which candivide and differentiate, or muscles cells from other sources, includingsmooth and skeletal muscles cells, to assist the heart to repair orreverse tissue damage, restore function, or to at least halt the damagecycle leading to further loss of healthy heart tissue. In addition manycirculatory diseases and injuries involve chronic or acute damage to, ornecrosis of, circulatory tissues, and the cells of which such tissuesare comprised. Cell-based and cell-derived therapies are of interest forthese conditions.

There is thus a need in the art for cell-based, or cell-derivedtherapies which can aid in healing damaged heart or circulatory tissues,or which can result in the repair or replacement of such damage in apatient.

SUMMARY OF THE INVENTION

The invention, in one of its aspects is generally directed to isolatedpostpartum-derived cells which are derived from postpartum tissue whichis substantially free of blood and which is capable of self-renewal andexpansion in culture and having the potential to differentiate alongmesenchymal lineage, towards cardiomyogenic, angiogenic and vasculogenicphenotypes, and further towards cells such as cardiomyocytes,endothelial cells, myocardial cells, epicardial cells, vascularendothelial cells, smooth muscle cells (e.g. vascular smooth musclecells), as well as cells of the excitatory and conductive systems, andprogenitors or more primitive relatives of the foregoing. The invention,in other aspects, is directed to populations of cells comprising suchcells, and therapeutic cell compositions, as well as methods of usingthe populations of cells for therapeutic treatment of cardiac orcirculatory damage or disease

In a first aspect, the invention provides isolated postpartum-derivedcells comprising L-valine-requiring cells derived from postpartum tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture and have the potential to differentiate into cellsof other phenotypes; for example cardiomyocytes, or their progenitors.The cells are capable of growth in atmospheres containing oxygen fromabout 5% to at least about 20% and comprise at least one of thefollowing characteristics:

-   -   the potential for at least about 40 doublings in culture;    -   attachment and expansion on a coated or uncoated tissue culture        vessel, wherein a coated tissue culture vessel comprises a        coating of gelatin, laminin, collagen, polyornithine,        vitronectin, or fibronectin;    -   production of at least one of tissue factor, vimentin, and        alpha-smooth muscle actin;    -   production of at least one of CD10, CD13, CD44, CD73, CD90,        PDGFr-alpha, PD-L2 and HLA-A,B,C;    -   lack of production of at least one of CD31, CD34, CD45, CD80,        CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as        detected by flow cytometry;    -   expression of at least one of interleukin 8; reticulon 1;        chemokine (C-X-C motif) ligand 1 (melonoma growth stimulating        activity, alpha); chemokine (C-X-C motif) ligand 6 (granulocyte        chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and        tumor necrosis factor, alpha-induced protein 3;    -   expression of at least one of C-type (calcium dependent,        carbohydrate-recognition domain) lectin, superfamily member 2        (activation-induced); Wilms tumor 1; aldehyde dehydrogenase 1        family, member A2; and renin; oxidized low density lipoprotein        (lectin-like) receptor 1; Homo sapiens, clone IMAGE:4179671,        mRNA, partial cds; protein kinase C, zeta; hypothetical protein        DKFZp564F013; downregulated in ovarian cancer 1; Homo sapiens        mRNA; cDNA DKFZp547K1113 (from clone DKFZp547K1113);    -   expression, which relative to a human cell that is a fibroblast,        a mesenchymal stem cell, or an ileac crest bone marrow cell, is        reduced for at least one of: short stature homeobox 2; heat        shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12        (stromal cell-derived factor 1); elastin (supravalvular aortic        stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA        DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2        (growth arrest-specific homeobox); sine oculis homeobox homolog        1 (Drosophila); crystallin, alpha B; dishevelled associated        activator of morphogenesis 2; DKFZP586B2420 protein; similar to        neuralin 1; tetranectin (plasminogen binding protein); src        homology three (SH3) and cysteine rich domain; B-cell        translocation gene 1, anti-proliferative; cholesterol        25-hydroxylase; runt-related transcription factor 3;        hypothetical protein FLJ23191; interleukin 11 receptor, alpha;        procollagen C-endopeptidase enhancer; frizzled homolog 7        (Drosophila); hypothetical gene BC008967; collagen, type VIII,        alpha 1; tenascin C (hexabrachion); iroquois homeobox protein 5;        hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;        Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744; cytokine        receptor-like factor 1; potassium intermediate/small conductance        calcium-activated channel, subfamily N, member 4; integrin,        alpha 7; DKFZP586L151 protein; transcriptional co-activator with        PDZ-binding motif (TAZ); sine oculis homeobox homolog 2        (Drosophila); KIAA1034 protein; early growth response 3;        distal-less homeobox 5; hypothetical protein FLJ20373; aldo-keto        reductase family 1, member C3 (3-alpha hydroxysteroid        dehydrogenase, type II); biglycan; fibronectin 1; proenkephalin;        integrin, beta-like 1 (with EGF-like repeat domains); Homo        sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422;        EphA3; KIAA0367 protein; natriuretic peptide receptor        C/guanylate cyclase C (atrionatriuretic peptide receptor C);        hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA        DKFZp564B222 (from clone DKFZp564B222); vesicle-associated        membrane protein 5 (myobrevin); EGF-containing fibulin-like        extracellular matrix protein 1; BCL2/adenovirus E1B 19 kDa        interacting protein 3-like; AE binding protein 1; cytochrome c        oxidase subunit VIIa polypeptide 1 (muscle); neuroblastoma,        suppression of tumorigenicity 1; insulin-like growth factor        binding protein 2, 36 kDa;    -   secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF,        FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; and    -   lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb,        MIP1b, I309, MDC, and VEGF, as detected by ELISA.

In certain embodiments, the postpartum-derived cell is anumbilicus-derived cell. In other embodiments it is a placenta-derivedcell. In specific embodiments, the cell has all identifying features ofany one of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074);cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); cell type PLA071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7)(ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCCAccession No. PTA-6068).

The postpartum-derived cells are preferably isolated in the presence ofone or more enzyme activities such as metalloprotease activity, neutralprotease activity and mucolytic enzyme activity. The cells preferablycomprise a normal karyotype, which is maintained as the cells arepassaged.

Preferred cells produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

In another of its several aspects, the invention provides populations ofcells comprising the cells described above. In certain embodiments thepopulations are incubated in the presence of one or more factors whichstimulate stem cell differentiation along a cardiogenic pathway orlineage. In other embodiments, cells are incubated in the presence ofcompounds which tend to stimulate differentiation along angiogenic,hemangiogenic, and vasculogenic pathways, or towards more committedcells such as cardiomyocytes, endothelial cells, myocardial cells,epicardial cells, vascular endothelial cells, smooth muscle cells (e.g.vascular smooth muscle cells), as well as cells of the excitatory andconductive systems, and progenitors of any of the foregoing, forexample, myoblasts, cardiomyoblasts, hemangioblasts, angioblasts and thelike or their progenitors. The populations can be providedtherapeutically to a patient, for example with heart or circulatorydisease, such as a cardiomyopathy, or with a cardiac injury, such asfrom myocardial infarction, or with any damage or disease of the heartor circulatory system. In presently preferred embodiments, thepopulation comprises about 50% postpartum-derived cells, while in otherpreferred embodiments the population is a substantially homogeneouspopulation of postpartum-derived cells.

The invention provides in another of its aspects therapeutic cellcompositions comprising a pharmaceutically-acceptable carrier andpostpartum-derived cells derived from human postpartum tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture and have the potential to differentiate into cellsof other phenotypes; for example smooth or skeletal muscles phenotypes.In preferred embodiments, cells can differentiate along pathways leadingto phenotypes of cardiomyocytes, cells of the endothelium, myocardium,epicardium, vascular endothelium, as well as smooth muscle cells (e.g.vascular smooth muscle cells), and cells of the excitatory andconductive systems (e.g. Purkinje cells), and progenitors of theforegoing. The cells are capable of growth in an atmosphere containingoxygen from about 5% to at least about 20%. The cells also requireL-valine for growth; have the potential for at least about 40 doublingsin culture; attach and expand on a coated or uncoated tissue culturevessel, wherein a coated tissue culture vessel is coated with gelatin,laminin, or fibronectin; produce tissue factor, vimentin, andalpha-smooth muscle actin; produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C; and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

The therapeutic cell compositions provided can be providedtherapeutically in a patient with a cardiomyopathy, or other heartdisease, or a cardiac injury, or any disease of the circulatory systemwhich, for example, involves cell injury or tissue/cell necrosis. Incertain embodiments, the therapeutic cell compositions comprise cellsinduced to differentiate along a cardiogenic, angiogenic, hemangiogenic,or vasculogenic pathway or lineage. The therapeutic cell compositionscan comprise cells or cellular products that stimulate adult stem cellsnaturally present in the heart, blood, blood vessels or the like todivide, or differentiate, or both. Preferably the therapeutic cellcompositions can at least survive, grow, stimulate in growth, stimulateangiogenesis or vasculogenesis, but in certain cases even nonviablecells such as senescent cells will be therapeutic, and in someembodiments, even dead cells, or fractions thereof will providetherapeutic improvements for a patient.

The therapeutic cell compositions are provided, for example, byinjection. In certain preferred embodiments, the therapeutic cellcompositions are provided by intracardiac injection. In otherembodiments, they are placed adjacent ot an inner or outer aspect of thecardiac muscles directly, or on a matrix as a cell-matrix complex. Thetherapeutic cell compositions provided in the form of a matrix-cellcomplex comprise matrices including, for example, biocompatiblescaffolds, lattices, self-assembling structures and the like, whetherbioabsorbable or not, liquid or solid. Many such matrices are known tothose of skill in the arts of tissue engineering, wound healing, and thelike. Thereapeutic compositions can also comprise additional biologicalcompounds or pharamaceuticals that may improve the therapeutic value tothe patient, and the compositions can also comprise one or moreadditional cells or cell types.

The therapeutic cell compositions, in certain embodiments also comprisecells that:

-   -   express at least one of interleukin 8; reticulon 1; chemokine        (C-X-C motif) ligand 1 (melonoma growth stimulating activity,        alpha); chemokine (C-X-C motif) ligand 6 (granulocyte        chemotactic protein 2); chemokine (C-X-C motif) ligand 3; and        tumor necrosis factor, alpha-induced protein 3;    -   express at least one of C-type (calcium dependent,        carbohydrate-recognition domain) lectin, superfamily member 2        (activation-induced); Wilms tumor 1; aldehyde dehydrogenase 1        family, member A2; and renin; oxidized low density lipoprotein        (lectin-like) receptor 1; Homo sapiens, clone IMAGE:4179671,        mRNA, partial cds; protein kinase C, zeta; hypothetical protein        DKFZp564F013; downregulated in ovarian cancer 1; Homo sapiens        mRNA; and cDNA DKFZp547K1113 (from clone DKFZp547K1113);    -   have reduced expression, relative to a human cell that is a        fibroblast, a mesenchymal stem cell, or an ileac crest bone        marrow cell, for at least one of: short stature homeobox 2; heat        shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12        (stromal cell-derived factor 1); elastin (supravalvular aortic        stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNA        DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2        (growth arrest-specific homeobox); sine oculis homeobox homolog        1 (Drosophila); crystallin, alpha B; dishevelled associated        activator of morphogenesis 2; DKFZP586B2420 protein; similar to        neuralin 1; tetranectin (plasminogen binding protein); src        homology three (SH3) and cysteine rich domain; B-cell        translocation gene 1, anti-proliferative; cholesterol        25-hydroxylase; runt-related transcription factor 3;        hypothetical protein FLJ23191; interleukin 11 receptor, alpha;        procollagen C-endopeptidase enhancer; frizzled homolog 7        (Drosophila); hypothetical gene BC008967; collagen, type VIII,        alpha 1; tenascin C (hexabrachion); iroquois homeobox protein 5;        hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;        Homo sapiens cDNA FLJ12280 fis, clone MAMMA1001744; cytokine        receptor-like factor 1; potassium intermediate/small conductance        calcium-activated channel, subfamily N, member 4; integrin,        alpha 7; DKFZP586L151 protein; transcriptional co-activator with        PDZ-binding motif (TAZ); sine oculis homeobox homolog 2        (Drosophila); KIAA1034 protein; early growth response 3;        distal-less homeobox 5; hypothetical protein FLJ20373; aldo-keto        reductase family 1, member C3 (3-alpha hydroxysteroid        dehydrogenase, type II); biglycan; fibronectin 1; proenkephalin;        integrin, beta-like 1 (with EGF-like repeat domains); Homo        sapiens mRNA full length insert cDNA clone EUROIMAGE 1968422;        EphA3; KIAA0367 protein; natriuretic peptide receptor        C/guanylate cyclase C (atrionatriuretic peptide receptor C);        hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA        DKFZp564B222 (from clone DKFZp564B222); vesicle-associated        membrane protein 5 (myobrevin); EGF-containing fibulin-like        extracellular matrix protein 1; BCL2/adenovirus E1B 19 kDa        interacting protein 3-like; AE binding protein 1; cytochrome c        oxidase subunit VIIa polypeptide 1 (muscle); neuroblastoma,        suppression of tumorigenicity 1; insulin-like growth factor        binding protein 2, 36 kDa;    -   secrete at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF,        HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; and    -   do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1b,        I309, MDC, and VEGF, as detected by ELISA.

In yet another of its aspects, the invention provides methods fortreating a patient with a disease or injury of the heart or circulatorysystem comprising administering a therapeutic postpartum-derived cellcomposition to a patient with a disease or injury of the heart orcirculatory system; and evaluating the patient for improvements, forexample in cardiac function. In certain preferred embodiments thedisease is a cardiomyopathy, either idiopathic or with a known cause,and either ischemic or nonischemic in nature.

Measurement of improvement include any means known in the art, butpreferred improvements include improvements in hemodynamic measurementsincluding but not limited to chest cardiac output (CO), cardiac index(CI), pulmonary artery wedge pressures (PAWP), and cardiac index (CI), %fractional shortening (% FS), ejection fraction (EF), left ventricularejection fraction (LVEF); left ventricular end diastolic diameter(LVEDD), left ventricular end systolic diameter (LVESD), contractility(e.g. dP/dt), pressure-volume loops, measurements of cardiac work, aswell as an increase in atrial or ventricular functioning; an increase inpumping efficiency, a decrease in the rate of loss of pumpingefficiency, a decrease in loss of hemodynamic functioning; and adecrease in complications associated with cardiomyopathy.

In some presently preferred embodiments, the method comprises inducingthe therapeutic postpartum-derived cells to differentiate along acardiogenic, angiogenic, hemangiogenic, or vasculogenic pathway or or atowards cells or progenitors of cells such as cardiomyocytes,endothelial cells, myocardial cells, epicardial cells, vascularendothelial cells, smooth muscle cells (e.g. vascular smooth musclecells), as well as cells of the excitatory and conductive systems. Cellswhich differentiate towards myoblasts, cardiomyoblasts, angioblasts,hemangioblasts, and the like are contemplated for use herein.

The administering is preferably in vivo by transplanting, grafting,implanting, injecting, fusing, delivering via catheter, or providing asa matrix-cell complex, or any other means known in the art for providingcell therapy. For many applications, the cells can simply be injected,for example intravenously, and the cells will locate or home inaccordance with their commitment in the direction of a phenotypeassociated with a particular tissue. For example, cells differentiatedin a cardiomyogenic lineage may home in on the cardiac muscle wheninjected intravenously anywhere in the body.

The invention also provides in another aspect, methods for treating apatient with a disease or injury of the heart or circulatory systemcomprising administering a therapeutic postpartum-derived cellcomposition to a patient with a disease or injury of the heart orcirculatory system; and evaluating the patient for improvements incardiac function, wherein the administering is with a population ofanother cell type. Administration of cocultures, mixed populations orother nonclonal populations are sometimes preferred. Other cell typeswhich can be coadministered are stem cells in certain embodiments, whilein others myoblasts, myocytes, cardiomyoblasts, cardiomyocytes,angioblasts, hemangioblasts or a progenitors of myoblasts, myocytes,cardiomyoblasts, angioblasts, hemangioblasts, or cardiomyocytes areused. Other angiogenic, hemangiogenic, and vasculogenic cells, or morecommited cells from such lineages are also suitable forcoadministration.

Also provided herein are methods for treating a patient with a diseaseor injury of the heart or circulatory system comprising administering atherapeutic postpartum-derived cell composition to a patient with adisease or injury of the heart or circulatory system; and evaluating thepatient for improvements in the diseased or injured state, wherein thetherapeutic cell compostion is administered as a matrix-cell complex. Incertain embodiments, the matrix is a scaffold, preferably bioabsorbable,comprising in addition to the matrix proper, at least thepostpartum-derived cells.

Co-cultures comprising the cells or cultures of the invention with othermammalian cells are also provided herein. Preferably these co-culturescomprise another mammalian cell line whose growth or therapeuticpotential, for example, is improved by the presence of theumbilicus-derived cells. Human cell lines are particular preferred. Suchco-cultures are useful for therapeutic application in vitro or in vivo.

Also provided herein are therapeutic compositions comprising apostpartum-derived cell and another therapeutic agent, factor, orbioactive agent, such as a pharmaceutical compound. Such bioactiveagents include, but are not limited to, IGF, LIF, PDGF, EGF, FGF, aswell as antithrombogenic, antiapoptotic agents, anti-inflammatoryagents, immunosuppressive or immunomodulatory agents, and antioxidants.Such therapeutic compositions can further comprise one or moreadditional cell types in addition to the PPDCs and the bioactivecomponent.

In addition to the above, compositions derived from the cells areprovided herein. Cell lysates, soluble cell fractions andmembrane-enriched cell fractions are provided herein. Extracellularmatrices derived from the cells, for example, fractions comprisingbasement membranes are also useful and are provided herein.

Compositons of the invention also include conditioned culture media asprovided herein. Such media have first been used to grow the cells orcultures of the invention, which during growth secrete one or moreuseful products into the medium. Conditioned medium from these novelcells are useful for many purposes, including for example, supportingthe growth of other mammalian cells in need of growth factors or trophicfactors secreted into the media by the cells and cultures of theinvention, and promoting, for example, angiogenesis.

Kits are also provided herein. Various preferred kits are thoseproviding the required components for practicing the several aspects ofthe inventions, for example kits for growth and maintenance of thecells, kits for isolation of the cells, kits for coculturing the cells,kits for utilizing the cells and culture in vitro and kits for utilizingthe cells and cultures in vivo are all provided herein. Kits are alsoprovided for the preparation of cell fractions derived from the cellsincluding for example, cell lysates, soluble and membrane-enrichedfractions, and extracellular fractions.

These and other aspects of the invention are described in more detailbelow.

DETAILED DESCRIPTION

Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An embryonic stem cell is a pluripotent cell from the inner cellmass of a blastocyst-stage embryo. A fetal stem cell is one thatoriginates from fetal tissues or membranes. A postpartum stem cell is amultipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the placenta andthe umbilical cord. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiatedcell is one that has taken on a more specialized (“committed”) positionwithin the lineage of a cell. The term committed, when applied to theprocess of differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.De-differentiation refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e. which cells it came from and what cells it can give rise to. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a mesodermal, ectodermalor endodermal lineage refers to a cell that becomes committed to aspecific mesodermal, ectodermal or endodermal lineage, respectively.Examples of cells that differentiate into a mesodermal lineage or giverise to specific mesodermal cells include, but are not limited to, cellsthat are adipogenic, chondrogenic, cardiogenic, dermatogenic,hematopoietic, hemangiogenic, myogenic, nephrogenic, urogenitogenic,osteogenic, pericardiogenic, or stromal. Examples of cells thatdifferentiate into ectodermal lineage include, but are not limited toepidermal cells, neurogenic cells, and neurogliagenic cells. Examples ofcells that differentiate into endodermal lineage include, but are notlimited to, pleurigenic cells, hepatogenic cells, cells that give riseto the lining of the intestine, and cells that give rise to pancreogenicand splanchogenic cells.

The cells of the present invention are generally referred to asumbilicus-derived cells (or UDCs). They also may sometimes be referredto more generally herein as postpartum-derived cells or postpartum cells(PPDCs). In addition, the cells may be described as being stem orprogenitor cells, the latter term being used in the broad sense. Theterm derived is used to indicate that the cells have been obtained fromtheir biological source and grown or otherwise manipulated in vitro(e.g., cultured in a Growth Medium to expand the population and/or toproduce a cell line). The in vitro manipulations of umbilical stem cellsand the unique features of the umbilicus-derived cells of the presentinvention are described in detail below.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture” or “cultured”). A primary cellculture is a culture of cells, tissues, or organs taken directly from anorganism(s) before the first subculture. Cells are expanded in culturewhen they are placed in a Growth Medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium,growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. When cells are cultured ina medium, they may secrete cellular factors that can provide trophicsupport to other cells. Such trophic factors include, but are notlimited to hormones, cytokines, extracellular matrix (ECM), proteins,vesicles, antibodies, and granules. The medium containing the cellularfactors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, proliferation and/or maturation of a cell, orstimulates increased activity of a cell.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

As used herein, the term Growth Medium generally refers to a mediumsufficient for the culturing of umbilicus-derived cells. In particular,one presently preferred medium for the culturing of the cells of theinvention in comprises Dulbecco's Modified Essential Media (alsoabbreviated DMEM herein). Particularly preferred is DMEM-low glucose(also DMEM-LG herein) (Invitrogen, Carlsbad, Calif.). The DMEM-lowglucose is preferably supplemented with serum, most preferably fetalbovine serum or human serum. Typically, 15% (v/v) fetal bovine serum(e.g. defined fetal bovine serum, Hyclone, Logan Utah) is added, alongwith antibiotics/antimycotics ((preferably 100 Unit/milliliterpenicillin, 100 milligrams/milliliter streptomycin, and 0.25microgram/milliliter amphotericin B; Invitrogen, Carlsbad, Calif.)), and0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). In some casesdifferent growth media are used, or different supplementations areprovided, and these are normally indicated in the text assupplementations to Growth Medium. In certain chemically-defined mediathe cells may be grown without serum present at all. In such cases, thecells may require certain growth factors, which can be added to themedium to support and sustain the cells. Presently preferred factors tobe added for growth on serum-free media include one or more of bFGF,EGF, IGF-I, and PDGF. In more preferred embodiments, two, three or allfour of the factors are add to serum free or chemically defined media.In other embodiments, LIF is added to serum-free medium to support orimprove growth of the cells.

Also relating to the present invention, the term standard growthconditions, as used herein refers to culturing of cells at 37° C., in astandard atmosphere comprising 5% CO₂. Relative humidity is maintainedat about 100%. While the foregoing conditions are useful for culturing,it is to be understood that such conditions are capable of being variedby the skilled artisan who will appreciate the options available in theart for culturing cells.

The following abbreviations are used herein:

-   -   ANG2 (or Ang2) for angiopoietin 2;    -   APC for antigen-presenting cells;    -   BDNF for brain-derived neurotrophic factor;    -   bFGF for basic fibroblast growth factor;    -   bid (BID) for “bis in die” (twice per day);    -   BSP for bone sialoprotein;    -   C:D for collagenase and dispase treatment    -   C:D:H for collagenase, dispase, and hyaluronidase treatment    -   CK18 for cytokeratin 18;    -   CXC ligand 3 for chemokine receptor ligand 3;    -   DAPI for 4′-6-Diamidino-2-phenylindole-2HCl    -   DMEM for Dulbecco's Minimal Essential Medium;    -   DMEM:lg (or DMEM.Lg, DMEM:LG) for DMEM with low glucose;    -   EDTA for ethylene diamine tetraacetic acid;    -   EGF for epidermal growth factor;    -   FACS for fluorescent activated cell sorting;    -   FBS for fetal bovine serum;    -   GCP-2 for granulocyte chemotactic protein 2;    -   GCP-2 for granulocyte chemotactic protein-2;    -   GFAP for glial fibrillary acidic protein;    -   HB-EGF for heparin-binding epidermal growth factor;    -   HCAEC for Human coronary artery endothelial cells;    -   HGF for hepatocyte growth factor;    -   hMSC for Human mesenchymal stem cells;    -   HNF-1 alpha for hepatocyte-specific transcription factor;    -   HUVEC for Human umbilical vein endothelial cells;

I309for a chemokine and the ligand for the CCR8 receptor, responsiblefor chemoattraction of TH2 type T-cells. I309 binds to endothelialcells, stimulates chemotaxis and invasion of these cells, and enhancesHUVEC differentiation into capillary-like structures in an in vitroMatrigel assay. Furthermore, I309is an inducer of angiogenesis in vivoin both the rabbit cornea and the chick chorioallantoic membrane assay(CAM).

-   -   IL-6 for interleukin-6;    -   IL-8 for interleukin-8;    -   K19 for keratin 19;    -   K8 for keratin 8;    -   KGF for keratinocyte growth factor;    -   MCP-1 for monocyte chemotactic protein 1;    -   MDC for macrophage-derived chemokine;    -   MIP1alpha for macrophage inflammatory protein 1alpha;    -   MIP1 beta for macrophage inflammatory protein 1beta;    -   MSC for mesenchymal stem cells;    -   NDRI for National Disease Research Interchange in Philadelphia,        Pa.    -   NHDF for Normal Human Dermal Fibroblasts;    -   NPE for Neural Progenitor Expansion media;    -   PBMC for Peripheral blood mononuclear cell;    -   PBS for phosphate buffered saline;    -   PDGFbb for platelet derived growth factor;    -   PDGFr/alpha for platelet derived growth factor receptor alpha;    -   PD-L2 for programmed—death ligand 2;    -   PO (or po) for “per os” (by mouth);    -   PPDC for postpartum-derived cell;    -   Rantes (or RANTES) for regulated on activation, normal T cell        expressed and secreted;    -   rhGDF-5 for recombinant human growth and differentiation factor        5;    -   SC for subcutaneously;    -   SDF-1 alpha for stromal-derived factor 1 alpha;    -   SHH for sonic hedgehog;    -   SOP for standard operating procedure;    -   TARC for thymus and activation-regulated chemokine;    -   TCP for Tissue culture plastic;    -   TGFbeta2 for transforming growth factor beta2;    -   TGFbeta-3 for transforming growth factor beta-3;    -   TIMP1 for tissue inhibitor of matrix metalloproteinase 1;    -   TPO for thrombopoietin;    -   TuJ1 for BIII Tubulin;    -   UDC for umbilicus-derived cell;    -   VEGF for vascular endothelial growth factor;    -   vWF for von Willebrand factor;    -   alphaFP for alpha-fetoprotein;

Description

Various articles and references are cited herein and throughout, each ofwhich is hereby incorporated in its entirety by such reference.

As summarized above, the invention, in one of its aspects is generallydirected to isolated postpartum-derived cells which are derived frompostpartum tissue which is substantially free of blood and which arecapable of self-renewal and expansion in culture and having thepotential to differentiate along mesenchymal lineage, towardscardiomyogenic, angiogenic and vasculogenic phenotypes, and furthertowards cells such as cardiomyocytes, endothelial cells, myocardialcells, epicardial cells, vascular endothelial cells, smooth muscle cells(e.g. vascular smooth muscle cells), as well as cells of the excitatoryand conductive systems, and progenitors of the foregoing. Other aspectsprovide populations comprising such cells, therapeutic cellcompositions, and methods of using the therapeutic cell compositions fortreatment of patients with injury or disease of the heart or circulatorysystem. The postpartum-derived cells have been characterized by theirgrowth properties in culture, by their cell surface markers, by theirgene expression, by their ability to produce certain biochemical trophicfactors, and by their immunological properties.

In a first aspect, the invention provides isolated postpartum-derivedcells comprising L-valine-requiring cells derived from mammalianpostpartum tissue substantially free of blood. The cells are capable ofself-renewal and expansion in culture and have the potential todifferentiate into cells of other phenotypes; for examplecardiomyocytes, or their progenitors. Cells may be isolated frompostpartum tissue, for example umbilicus or placenta, of any mammal ofinterest by the techniques provided herein. Human cells are presentlypreferred. The cells can be grown under a wide range of conditions,including a wide variety of culture media, and environmental conditions.The cells can be grown at least from about 35° C. to about 39° C., andpossibly a wider range depending on other conditions. The cells can begrown in chemically-defined media, or in medium with added mammalianserum, for example fetal bovine serum. The cells also toleratecryopreservation at various stages. Cells can maintained frozen, orbanked at temperatures preferably below −80° C. for long periods.Temperature below −90° C. are also preferred and can be attained byspecialized electric freezers. Temperature of −180° C. and below arealso preferred and can be attained in liquid- or vapor-phase nitrogen.Tissues can also be banked prior to the isolation of the cells.Preferably such tissues are banked within a few hours or less after thecompletion of the pregnancy.

The cells are capable of growth in atmospheres containing oxygen fromabout 5% to at least about 20% and comprise at least one of thefollowing characteristics: the cells have the potential for at leastabout 40 doublings in culture; the cells preferably are adherent, thusattachment and expansion on a coated or uncoated tissue culture vesselis preferred, wherein a coated tissue culture vessel comprises a coatingof gelatin, laminin, collagen, polyornithine, polylysine, vitronectin,or fibronectin. While the cells are preferably adherent and isolated assuch, the cells have been grown in a spherical form in some embodiments.

Many populations of cells are present in postpartum tissue, but thecells of the invention preferably produce of at least one of tissuefactor, vimentin, and alpha-smooth muscle actin; more preferred arecells which produce each of tissue factor, vimentin, and alpha-smoothmuscle actin; production of at least one of CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C is also preferred. The cells arealso characterized in their lack of production of at least one of CD31,CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, andHLA-DR,DP,DQ, as detected by flow cytometry; more preferable cells lackproduction of all of these surface markers. Also preferred are cellswhich express at least one of interleukin 8; reticulon 1; chemokine(C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha);chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2);chemokine (C-X-C motif) ligand 3; and tumor necrosis factor,alpha-induced protein 3. The cells, in other embodiments, preferablyalso express one or more of C-type (calcium dependent,carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced); Wilms tumor 1; aldehyde dehydrogenase 1 family,member A2; and renin; oxidized low density lipoprotein (lectin-like)receptor 1; Homo sapiens, clone IMAGE:4179671, mRNA, partial cds;protein kinase C, zeta; hypothetical protein DKFZp564F013; downregulatedin ovarian cancer 1; Homo sapiens mRNA; and cDNA DKFZp547K1113 (fromclone DKFZp547K1113). Preferred cells also have expression, whichrelative to a human cell that is a fibroblast, a mesenchymal stem cell,or an ileac crest bone marrow cell, is reduced for at least one of:short stature homeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-Cmotif) ligand 12 (stromal cell-derived factor 1); elastin (supravalvularaortic stenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNADKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2 (growtharrest-specific homeobox); sine oculis homeobox homolog 1 (Drosophila);crystallin, alpha B; dishevelled associated activator of morphogenesis2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin(plasminogen binding protein); src homology three (SH3) and cysteinerich domain; B-cell translocation gene 1, anti-proliferative;cholesterol 25-hydroxylase; runt-related transcription factor 3;hypothetical protein FLJ23191; interleukin 11 receptor, alpha;procollagen C-endopeptidase enhancer; frizzled homolog 7 (Drosophila);hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C(hexabrachion); iroquois homeobox protein 5; hephaestin; integrin, beta8; synaptic vesicle glycoprotein 2; Homo sapiens cDNA FLJ12280 fis,clone MAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, alpha 7; DKFZP586L151 protein; transcriptionalco-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog2 (Drosophila); KIAA1034 protein; early growth response 3; distal-lesshomeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-likerepeat domains); Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic peptide receptor C);hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222(from clone DKFZp564B222); vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa. The skilled artisan will appreciatethat the expression of a wide variety of genes is convenientlycharacterized on oligonucleotide arrays, for example on a AffymetrixGENECHIP.

The cells secrete a variety of biochemically active factors, such asgrowth factors, chemokines, cytokines and the like. Preferred cellssecrete at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF,BDNF, TPO, MIP1a, RANTES, and TIMP1; preferred cells may alternativelybe characterized in their lack of secretion of at least one ofTGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC, and VEGF, as detected byELISA. These and other characteristics are available to identify andcharacterize the cells, and distinguish the cells of the invention fromothers known in the art.

In preferred embodiments, the cell comprises two or more of theforegoing characteristics. More preferred are those cells comprising,three, four, or five or more of the characteristics. Still morepreferred are those isolated postpartum cells comprising six, seven, oreight or more of the characteristics. Still more preferred presently arethose cells comprising all nine of the claimed characteristics.

Also presently preferred are cells that produce at least two of tissuefactor, vimentin, and alpha-smooth muscle actin. More preferred arethose cells producing all three of the proteins tissue factor, vimentin,and alpha-smooth muscle actin.

The skilled artisan will appreciate that cell markers are subject tovary somewhat under vastly different growth conditions, and thatgenerally herein described are characterizations in Growth Medium, orvariations thereof. Postpartum-derived cells that produce of at leastone, two, three, or four of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C are preferred. More preferred are those cellsproducing five, six, or seven of these cell surface markers. Still morepreferred are postpartum cells that can produce all eight of theforegoing cell surface marker proteins.

Similarly, postpartum cells that lack of production of at least one,two, three, four of the proteins CD31, CD34, CD45, CD80, CD86, CD117,CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flowcytometry are presently preferred. Cells lacking production of at leastfive, six, seven or eight or more of these markers are also preferred.More preferred are cells which lack production of at least nine or tenof the cell surface markers. Most highly preferred are those cellslacking production of all eleven of the foregoing identifying proteins.

Presently preferred cells produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

Presently, it is preferred that postpartum-derived cells express atleast one, two or three of interleukin 8; reticulon 1; chemokine (C-X-Cmotif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine(C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C-X-C motif) ligand 3; and tumor necrosis factor, alpha-induced protein3. More preferred are those cells which express four or five, and stillmore preferred are cell capable of expressing all six of the foregoinggenes.

In some embodiments, the cells preferably also express two, three, fouror more of C-type (calcium dependent, carbohydrate-recognition domain)lectin, superfamily member 2 (activation-induced); Wilms tumor 1;aldehyde dehydrogenase 1 family, member A2; renin; oxidized low densitylipoprotein (lectin-like) receptor 1; Homo sapiens, clone IMAGE:4179671,mRNA, partial cds; protein kinase C, zeta; hypothetical proteinDKFZp564F013; down-regulated in ovarian cancer 1; Homo sapiens mRNA; andcDNA DKFZp547K1113 (from clone DKFZp547K1113). In other embodiments, itis preferred that the cells express five, six, seven or eight of theforegoing. Also preferred are those cells expressing genes correspondingto nine, ten or even all of the foregoing sequences.

For some embodiments, preferred are cells, which relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, have reduced expression for at least one of the genescorresponding to: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2 (growth arrest-specific homeobox); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; dishevelled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine -rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7 (Drosophila); hypothetical gene BC008967; collagen, type VIII, alpha1; tenascin C (hexabrachion); iroquois homeobox protein 5; hephaestin;integrin, beta 8; synaptic vesicle glycoprotein 2; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, alpha 7; DKFZP586L151 protein;transcriptional co-activator with PDZ-binding motif (TAZ); sine oculishomeobox homolog 2 (Drosophila); KIAA1034 protein; early growth response3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-ketoreductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase,type II); biglycan; fibronectin 1; proenkephalin; integrin, beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; Homo sapiens mRNA; cDNADKFZp564B222 (from clone DKFZp564B222); vesicle-associated membraneprotein 5 (myobrevin); EGF-containing fibulin-like extracellular matrixprotein 1; BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AEbinding protein 1; cytochrome c oxidase subunit VIIa polypeptide 1(muscle); neuroblastoma, suppression of tumorigenicity 1; insulin-likegrowth factor binding protein 2, 36 kDa. More preferred are cells thathave, relative to human fibroblasts, mesenchymal stem cells, or ileaccrest bone marrow cells, reduced expression of at least 5, 10, 15 or 20genes corresponding to those listed above. Presently more preferred arecell with reduced expression of at least 25, 30, or 35 of the genescorresponding to the listed sequences. Also more preferred are thosepostpartum-derived cells having expression that is reduced, relative tothat of a human fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, of genes corresponding to 35 or more, 40 or more, oreven all of the sequences listed.

Secretion of certain growth factors and other cellular proteins can makecells of the invention particularly useful. Preferred postpartum-derivedcells secrete at least one, two, three or four of MCP-1, IL-6, IL-8,GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. Cellswhich secrete more than five, six, seven or eight of the listed proteinsare also useful and preferred. Cells which can secrete at least nine,ten, eleven or more of the factors are more preferred, as are cellswhich can secrete twelve or more, or even all thirteen of the proteinsin the foregoing list.

While secretion of such factors is useful, cells can also becharacterized by their lack of secretion of factors into the medium.Postpartum-derived cells that lack secretion of at least one, two, threeor four of TGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC, and VEGF, asdetected by ELISA, are presently preferred for use. Cells that arecharacterized in their lack secretion of five or six of the foregoingproteins are more preferred. Cells which lack secretion of all seven ofthe factors listed above are also preferred.

The postpartum-derived cells are preferably isolated in the presence ofone or more enzyme activities. A broad range of digestive enzymes foruse in cell isolation from tissue are known in the art, includingenzymes ranging from those considered weakly digestive (e.g.deoxyribonucleases and the neutral protease, dispase) to stronglydigestive (e.g. papain and trypsin). For example, collagenases are knownto be useful for isolating various cells from (tissues.Deoxyribonucleases can digest single-stranded DNA and can minimizecell-clumping during isolation. Enzymes can be used alone or incombination. Serine protease are preferably used in a sequence followingthe use of other enzymes as they may degrade the other enzymes beingused. The temperature and time of contact with serine proteases must bemonitored. Serine proteases may be inhibited with alpha 2 microglobulinin serum and therefore the medium used for digestion is preferablyserum-free. EDTA and DNase are commonly used and may improve yields orefficiencies. Preferred methods involve enzymatic treatment with forexample collagenase and dispase, or collagenase, dispase, andhyaluronidase, and such methods are provided wherein in certainpreferred embodiments, a mixture of collagenase and the neutral proteasedispase are used in the dissociating step. Presently preferred aremucolytic enzyme activities, metalloproteases, neutral proteases, serineproteases (such as trypsin, chymotrypsin, or elastase), anddeoxyribonucleases. More preferred are enzyme activites selected frommetalloproteases, neutral proteases and mucolytic activities. Presentlypreferred are cells that are isolated in the presence of one or moreactivities of collagenase, hyaluronidase and dispase. More preferred arethose cells isolated in the presence of a collagenase from Clostridiumhistolyticum, and either of the protease activities, dispase andthermolysin. Still more preferred are cells isolated with collagenaseand dispase enzyme activities. Also preferred are such cells isolated inthe presence of a hyaluronidase activity, in addition to collagenase anddispase activity. The skilled artisan will appreciate that many suchenzyme treatments are known in the art for isolating cells from varioustissue sources. Also useful for isolation of certain cells of theinvention are commercial enzyme preparations such as blends of enzymes,for example, LIBERASE Blendzymes available from Roche Diagnostics. Theskilled artisan will appreciate the methods for optimizing enzyme useduring isolation, and information on such optimization procedures isavailable from the manufacturers of commercial enzymes. Preferred arethose methods which can result in homogenous populations or nearlyhomogeneous populations of cells.

The cells also preferably comprise a normal karyotype, which ismaintained as the cells are passaged. Karyotyping is particularly usefulfor identifying and distinguishing neonatal from maternal cells derivedfrom placenta. Methods for karyotyping are available and known to thoseof skill in the art.

Among cells that are presently preferred for use with the invention inseveral of its aspects are postpartum cells having the characteristicsdescribed above and more particularly those wherein the cells havenormal karyotypes and maintain normal karyotypes with passaging, andduring cryopreservation and subsequent thawing and use, and furtherwherein the cells express each of the markers CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, and HLA-A,B,C, wherein the cells produce theimmunologically-detectable proteins which correspond to the listedmarkers. Still more preferred are those cells which in addition to theforegoing do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD 117, CD141, or HLA-DR,DP,DQ, as detected by flowcytometry.

Certain prior art cells having the potential to differentiate alonglines leading to various phenotypes are unstable and thus canspontaneously differentiate. Presently preferred for use with theinvention are cells which do not spontaneously differentiate, forexample along cardiomyogenic, angiogenic, hemangiogenic, orvascualogenic lines. Preferred cells when grown in Growth Medium aresubstantially stable with respect to the cell markers produced on theirsurface, and with respect to the expression pattern of various genes,for example as determined using oligonucleotide arrays, such as anAffymetrix GENECHIP. The cells remain substantially constant in theirbiochemical, genetic, and immunological characteristics, for example,their cell surface markers, over passaging, and through multiplepopulation doublings.

In another of its several aspects, the invention provides populations ofcells comprising the cells described above. Cell populations are usefulin connection with the methods of the invention, as well as inconnection with making the therapeutic cell compositions and celllysates in larger amounts than isolated cells can provide.

Preferred populations comprise from about 1% postpartum-derived cells toabout 10% postpartum cells. More preferred populations comprise at leastabout 10% postpartum-derived cells. More preferred populations compriseat least about 25% postpartum-derived cells. Also, some preferredpopulations comprise about 50% postpartum-derived cells. Suchpopulations may be useful for coculture or other cultures wherein thecells are equally populous and divide at the same rate, or where thepopulation is adjusted to about 50% of each culture after expansion ofthe cultures in coculture or separately. More preferred for someapplications are populations comprising at least about 65%postpartum-derived cells. Populations that comprising at least 90%postpartum-derived cells are highly preferred for certain aspects of theinvention. More preferred populations comprise substantially onlypostpartum-derived cells.

The populations may comprise a clonal cell line of postpartum-derivedcells. Such populations are particularly useful wherein a cell clonewith highly desirable functionality is isolated. Both neotal andmaternal clones are useful and are provided herein. Methods of isolatingclonal cell lines from cultured cells are known in the art.

The invention also provides cell lysates, soluble cell fractions andmembrane-enriched cell fractions prepared from the populations of thepostpartum cells. Such lysates and fractions have many utilities. Use ofcell lysates, and more particularly soluble cell fractions, in vivoallows the beneficial intracellular milieu to be used in a patientallogeneic patient without stimulating allogeneic lymphocytes, orgenerating other adverse immunological responses, or triggeringrejection. Methods of lysing cells are well-known in the art and includevarious means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their Growth Medium and thus containingsecreted growth factors and the like, or may be prepared from cellswashed free of medium in, for example, PBS or another solution. Formaking lysates from cells directly in the growth medium it is preferredthat cells are grown in serum from the species in which the lysates areto be used, in some embodiments, washed cells may be preferred. Washedcells may be resuspended at concentrations greater than the originalpopulation density if preferred. Cell lysates prepared from populationsof postpartum-derived cells may be used as is, further concentrated, byfor example, ultrafiltration or lyophilization, or even dried, enriched,partially purified, combined with pharmaceutically-acceptable carriersor diluents as are known in the art, or combined with other compoundssuch as biologicals, for example pharmaceutically useful proteincompositions. Cell lysates may be used in vitro or in vivo, alone or,for example, with syngeneic or autologous live cells. The lysates, ifintroduced in vivo, may be introduced locally at a site of treatment, orremotely to provide, for example, needed cellular growth factors to apatient. Preferably, the lysates are not immunogenic, and morepreferably they are immunologically tolerated in a broad population ofsyngeneic and allogeneic recipients without adverse immunologicalconsequences or reaction. Cell lysates of the invention are useful fromcells at any stage or age which have been grown under conditions forgrowth and expansion, for example on Growth Medium. Even senescent cellsare useful for the preparation of lysate and can provide certain factorswhich are biologically useful. Nonviable or even dead or killed cellshave utility for preparing lysates, and cellular fractions. Also usefulare lysates from cells which have been exposed to factors which tend toinduce them along a mesenchymal pathway, particularly towardscardiomyogenic, angiogenic, hemangiogenic, and vasculogenic lines. Celllysates from differentiated cells, or cells more committed than thePPDCs are also desirable. For example, lysates from cells withcharacteristics of cardiomyoblasts, cardiomyocytes, angioblasts,hemangioblasts and the like, or their progenitors are also useful andcontemplated for use herewith.

Also provided herein are populations of cells incubated in the presenceof one or more factors which stimulate stem cell differentiation along acardiogenic, angiogenic, hemangiogenic, or vasculogenic pathway. Suchfactors are known in the art and the skilled artisan will appreciatethat determination of suitable conditions for differentiation can beaccomplished with routine experimentation. Optimization of suchconditions can be accomplished by statistical experimental design andanalysis, for example, response surface methodology allows simultaneousoptimization of multiple variables, for example biological cultureconditions. Presently preferred factors include, but are not limited tofactors, such as growth factors, chemokines, cytokines, cellularproducts, demethylating agents, and other stimuli which are now known orlater determined to stimulate differentiation, for example of stemcells, along cardiogenic, angiogenic, hemangiogenic, or vasculogenicpathways or lineages. Presently preferred for inducing or stimulatingdifferentiation along a cardiogenic, angiogenic, hemangiogenic, orvasculogenic pathway are cells incubated in the presence of factorscomprising at least one of a demethylation agent, a member of BMP, FGF,TAK, GATA, Csx, NK, MEF2, ET-1, and Wnt factor families, Hedgehog,Csx/Nkx-2.5, and anti-Wnt factors. DNA methylation is known to silencecertain genes, preventing their expression, demethylation may allowexpression of such genes, including some required for differentiation.Preferred demethylation agents include inhibitors of DNAmethyltransferases or inhibitors of histone deacetylase, or inhibitorsof a repressor complex.

Presently preferred demethylation agents comprise at least one of5-azacytidine, 5-aza-2′-deoxycytidine, DMSO, chelerythrine chloride,retinoic acid or salts thereof, 2-amino-4-(ethylthio)butyric acid,procainamide, and procaine. Inclusion of such factors tend to induce thecells to differentiate along mesenchymal lines, toward a cardiomyogenicpathway, as determined, for example, by the expression of at least oneof cardiomyosin, skeletal myosin, or GATA4; or by the acquisition of abeating rhythm, spontaneous or otherwise induced; or by the ability tointegrate at least partially into a patient's cardiac muscle withoutinducing arrhythmias.

In preferred embodiments herein, cells induced with one or more factorsas identified above may become cardiomyogenic, angiogenic,hemangiogenic, or vasculogenic cells, or progenitors or primitiverelatives thereof. Preferably at least some of the cells can integrateat least partially into the patient's heart or vasculature tree,including but not limited to heart muscle, vascular and other structuresof the heart, blood vessels, and the like. More preferred aredifferentiated cells acquiring two or more of the indicia ofcardiomyogenic, cells or their progenitors, and able to fully integrateinto a patient's heart or vasculature. Still more preferred are thosecells which when placed into a patient, result in no increase inarrhythmias, heart defects, blood vessel defects or other anamoliesrelated to the patient's circulatory system or health. Also preferredare those cells which can stimulate stem cells naturally present in thepatient's cardiac muscle, blood vessels, blood and the like tothemselves differentiate into for example, cardiomyocytes, or at leastalong cardiomyogenic, angiogenic, hemangiogenic, or vasculogenic lines.Equally preferred are PPDCs which can support the stem cells naturallypresent in the patient's cardiac muscle, blood, blood stream and thelike to grow and expand and be available for later differentiation.

The populations can be provided therapeutically to a patient, forexample with a disease of the heart or circulatory system. Commonexamples, not intended to limit the invention, include congestive heartfailure due to atherosclerosis, cardiomyopathy, or cardiac injury, suchas from myocardial infarction or wound (acute or chronic). In presentlypreferred embodiments, the population comprises about 50%postpartum-derived cells, while in other preferred embodiments thepopulation is a substantially homogeneous population ofpostpartum-derived cells. In other embodiments the population comprisesat least about 1, 10, 20, 25, 30, 33, 40, 60, 66, 70, 75, 80, or 90%postpartum-derived cells.

Co-cultures comprising the cells or cultures of the invention with othermammalian cells are also provided herein. Preferably these co-culturescomprise another mammalian cell line whose growth or therapeuticpotential, for example, is improved by the presence of theumbilicus-derived cells. Human cell lines are particular preferred. Suchco-cultures are useful for therapeutic application in vitro or in vivo.

Also provided herein are therapeutic compositions comprising apostpartum-derived cell and another therapeutic agent, factor, orbioactive agent, such as a pharmaceutical compound. Such bioactiveagents include, but are not limited to, IGF, LIF, PDGF, EGF, FGF, aswell as antithrombogenic, antiapoptotic agents, anti-inflammatoryagents, immunosuppressive or immunomodulatory agents, and antioxidants.Such therapeutic compositions can further comprise one or moreadditional cell types in addition to the PPDCs and the bioactivecomponent.

Thus, in conjunction with therapeutic cells, other biologically activemolecules, such as antithrombogenic agents, anti-apoptotic agents, andanti-inflammatory agents may be useful and may be administered insequence with, or coadministered with the the cells, individually or incombinations or two or more such compounds or agents. For example,anti-apoptotic agents may be useful to minimize programmed cell death.Such agents include but are not limited to EPO, EPO derivatives andanalogs, and their salts, TPO, IGF-I, IGF-II, hepatocyte growth factor(HGF), and caspase inhibitors. Anti-inflammatory agents include but arenot limited to P38 MAP kinase inhibitors, statins, IL-6 and IL-1inhibitors, Pemirolast, Tranilast, Remicade, Sirolimus, nonsteroidalanti-inflammatory compounds, for example, Tepoxalin, Tolmetin, andSuprofen.

Other bioactive factors or therapeutic agents which can becoadministered with the therapeutic cells of the invention include, forexample, antithrombogenic factors, immunosuppressive or immunomodulatoryagents, and antioxidants. Examples of immunosuppressive and immudulatoryagents include calcineurin inhibitors, for example cyclosporine,Tacrolimus, mTOR inhibitors such as Sirolimus or Everolimus;anti-proliferatives such as azathioprine and mycophenolate mofetil;corticosteroids for example prednisolone or hydrocortisone; antibodiessuch as monoclonal anti-IL-2Rα receptor antibodies, Basiliximab,Daclizumab; polyclonal anti-T-cell antibodies such as anti-thymocyteglobulin (ATG), anti-lymphocyte globulin (ALG), and the monoclonalanti-T cell antibody OKT3. Antithrombogenic compounds which can betherapeutically provided in conjunction with the cells of the inventioninclude, for example, heparin, heparin derivatives, urokinase, and PPack(dextrophenylalanine proline arginine chloromethylketone); antithrombincompounds, platelet receptor antagonists, anti-thrombin antibodies,anti-platelet receptor antibodies, aspirin, dipyridamole, protamine,hirudin, prostaglandin inhibitors, and platelet inhibitors. Antioxidantsare well known in the art of course and any pharamacueitcally acceptableantioxidant may be administered in conjunction with the cells of theinvention including probucol; vitamins A, C, and E, coenzyme Q-10,glutathione, L cysteine, N-acetylcysteine, or antioxidant derivative,analogs or salts of the foregoing.

In addition to the above, compositions derived from the cells areprovided herein. Cell lysates, soluble cell fractions andmembrane-enriched cell fractions are provided herein, as described abovein detail. Extracellular matrices derived from the cells, for example,comprising basement membranes are also useful and are provided herein.Cell lysates, soluble cell fractions, membrane-enriched cell fractionsand extracellular matrix derived from the cells can all be administeredto patients as appropriate, or coadministered with the cells of theinvention, with or without additional cells or cell types.

Compositons of the invention also include conditioned culture media asprovided herein. Such media have first been used to grow the cells orcultures of the invention, which during growth secrete one or moreuseful products into the medium. Conditioned medium from these novelcells are useful for many purposes, including for example, supportingthe growth of other mammalian cells in need of growth factors or trophicfactors secreted into the media by the cells and cultures of theinvention, and promoting, for example, angiogenesis. Methods ofpreparing and storing conditioned media are known in the art andprimarily involve removal of the cells, for example by centrifugation.

The invention provides in another of its aspects therapeutic cellcompositions comprising a pharmaceutically-acceptable carrier andpostpartum-derived cells derived from mammalian postpartum tissuesubstantially free of blood. The cells are capable of self-renewal andexpansion in culture and have the potential to differentiate alongmesenchymal lineage, towards cardiomyogenic, angiogenic and vasculogenicphenotypes, and further towards cells such as cardiomyocytes,endothelial cells, myocardial cells, epicardial cells, vascularendothelial cells, smooth muscle cells (e.g. vascular smooth musclecells), as well as cells of the excitatory and conductive systems, andprogenitors of the foregoing. The cells are capable of growth in anatmosphere containing oxygen from about 5% to at least about 20%. Thecells also require L-valine for growth; have the potential for at leastabout 40 doublings in culture; attach and expand on a coated or uncoatedtissue culture vessel, wherein a coated tissue culture vessel is coatedwith gelatin, laminin, or fibronectin; produce tissue factor, vimentin,and alpha-smooth muscle actin; produce each of CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, and HLA-A,B,C; and do not produce any of CD31, CD34,CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry. Inpreferred embodiments the cells are derived from human tissue.

The therapeutic cell compositions provided can be providedtherapeutically in a patient with a disease of the heart or circulatorysystem, such as a cardiomyopathy, or other heart disease, or a cardiacinjury. In certain embodiments, the therapeutic cell compositionscomprise cells induced to differentiate along a cardiogenic, angiogenic,hemangiogenic, or vasculogenic pathway or lineage. The therapeutic cellcompositions can comprise cells or cell products that stimulate adultstem cells present in the heart, blood, blood vessels and the like, todivide, or differentiate, or both.

The therapeutic cell compositions are provided, for example, byinjection. In certain embodiments, the therapeutic cell compositions areprovided by intracardiac injection. In other embodiments, the injectionmay be onto the surface of the heart, into an adjacent area, or even toa more remote area. In preferred embodiments, the cells can home to thediseased or injured area. Particularly preferred are cells that can beinjected intravenously and locate appropriately to the desired site ofaction, for example, cardiomyocytes or their progenitors preferably havethe ability to locate and home to the heart muscle or it structures.

The therapeutic cell compositions can also be provided in the form of amatrix-cell complex. Matrices include biocompatible scaffolds, lattices,self-assembling structures and the like, whether bioabsorbable or not,liquid, gel, or solid. Such matrices are known in the arts oftherapeutic cell treatment, surgical repair, tissue engineering, andwound healing. Preferably the matrices are pretreated with thetherapeutic cells. More preferably the matrices are populated with cellsin close association to the matrix or its spaces. The cells can adhereto the matrix in some embodiments, in others the cells are entrapped orcontained within the matrix spaces. Most preferred are those matrix-cellcomplexes were the cells are growing in close association with thematrix and when used therapeutically, in growth of the patient's cellsis stimulated and supported, and proper angiogenesis is similarlystimulated or supported. The matrix-cell compositions can be introducedinto a patients body in any way known in the art, including but notlimited to implantation, injection, surgical attachment, transplantationwith other tissue, injection, and the like. In some embodiemtns, thematrices form in vivo, or even more preferably in situ, for example insitu polymerizable gels can be used in accordance with the invention.Examples of such gels are known in the art.

In some embodiments, the cells of the invention, or co-cultures thereof,may be seeded onto such three-dimensional matrices, such as scaffoldsand implanted in vivo, where the seeded cells may proliferate on or inthe framework or help establish replacement tissue in vivo with orwithout cooperation of other cells.

Growth of PPDCs or co-cultures thereof on the three-dimensionalframework preferably results in the formation of a three-dimensionaltissue, or foundation therefor, which can be utilized in vivo, forexample for repair of damaged or diseased tissue. For example, thethree-dimensional scaffolds can be used to form tubular structures, forexample for use in repair of blood vessels; or aspects of thecirculatory system or coronary structures.

In accordance with one aspect of the invention, PPDCs or co-culturesthereof are inoculated, or seeded on a three-dimensional framework ormatrix, such as a scaffold, a foam or hydrogel. The framework may beconfigured into various shapes such as generally flat, generallycylindrical or tubular, or can be completely free-form as may berequired or desired for the corrective structure under consideration. Insome embodiments, the PPDCs grow on the three dimensional structure,while in other embodiments, the cells only survive, or even die, howeverin doing so they stimulate or promote ingrowth of new tissue, forexample, and preferably vascularization. PPDCs may be co-administeredwith myocytes, myoblasts, vascular endothelial cells, dermalfibroblasts, keratinocytes, and other soft tissue type progenitors,including stem cells. When grown in this three-dimensional system, theproliferating cells mature and segregate properly to form components ofadult tissues analogous to counterparts found naturally in vivo.

For example, but not by way of limitation, the matrix may be designedsuch that the matrix structure: (1) supports the PPDCs or co-culturesthereof without subsequent degradation; (2) supports the PPDCs orco-cultures thereof from the time of seeding until the tissue transplantis remodeled by the host tissue; (2) allows the seeded cells to attach,proliferate, and develop into a tissue structure having sufficientmechanical integrity to support itself in vitro, at which point, thematrix is degraded. A review of matrix design is provided by Hutmacher,J. Biomat. Sci. Polymer Edn., 12(1):107-124 (2001).

The matrices, scaffolds, foams and self-assembling systems contemplatedfor use herein can be implanted in combination with any one or morecells, growth factors, drugs, or other components, such as bioactiveagents that promote healing, or in growth of tissue, or stimulatevascularization or innervation thereof or otherwise enhance or improvethe therapeutic outcome or the practice of the invention, in addition tothe cells of the invention.

The cells of the invention can be grown freely in culture, removed fromthe culture and inoculated onto a three-dimensional framework.Inoculation of the three-dimensional framework with a concentration ofcells, e.g., approximately 10⁶ to 5×10⁷ cells per milliliter, preferablyresults in the establishment of the three-dimensional support inrelatively shorter periods of time. Moreover in some application it maybe preferably to use a greater or lesser number of cells depending onthe result desired.

In some embodiments, it is useful to re-create in culture the cellularmicroenvironment found in vivo, such that the extent to which the cellsare grown prior to implantation in vivo or use in vitro may vary. PPDCsor co-cultures thereof may be inoculated onto the framework before orafter forming the shape desired for implantation, e.g., ropes, tubes,filaments, and the like. Following inoculation of the cells onto theframework, the framework is preferably incubated in an appropriategrowth medium. During the incubation period, the inoculated cells willgrow and envelop the framework and may for example bridge, or partiallybridge any interstitial spaces therein. It is preferable, but notrequired to grow the cells to an appropriate degree which reflects thein vivo cell density of the tissue being repaired or regenerated. Inother embodiments, the presence of the PPDCs, even in relatively lownumbers on the framework encourages ingrowth of the other healthy cellsto facilitate healing for example of a wounded or necrotic tissue.

Examples of matrices, for example scaffolds which may be used foraspects of the invention include mats (woven, knitted, and morepreferably nonwoven) porous or semiporous foams, self assemblingpeptides and the like. Nonwoven mats may, for example, be formed usingfibers comprised of natural or synthetic polymers. In a preferredembodiment, absorbable copolymers of glycolic and lactic acids(PGA/PLA), sold under the tradename VICRYL (Ethicon, Inc., Somerville,N.J.) are used to form a mat. Foams, composed of, for example,poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,formed by processes such as freeze-drying, or lyophilization, asdiscussed in U.S. Pat. No. 6,355,699, can also serve as scaffolds. Gelsalso form suitable matrices, as used herein. Examples include in situpolymerizable gels, and hydrogels, for example composed ofself-assembling peptides. These materials are frequently used assupports for growth of tissue. In situ-forming degradable networks arealso suitable for use in the invention (see, e.g., Anseth, K. S. et al.,2002, J. Controlled Release 78: 199-209; Wang, D. et al., 2003,Biomaterials 24: 3969-3980; U.S. Patent Publication 2002/0022676 to Heet al.). These materials are formulated as fluids suitable forinjection, then may be induced by a variety of means (e.g., change intemperature, pH, exposure to light) to form degradable hydrogel networksin situ or in vivo.

According to a preferred embodiment, the framework is a felt, which canbe composed of a multifilament yarn made from a bioabsorbable material,e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarnis made into a felt using standard textile processing techniquesconsisting of crimping, cutting, carding and needling. In anotherpreferred embodiment the cells of the invention are seeded onto foamscaffolds that may be composite structures. In addition, thethree-dimensional framework may be molded into a useful shape, such as aspecific structure in the body to be repaired, replaced, or augmented.

The framework may be treated prior to inoculation of the cells of theinvention in order to enhance cell attachment. For example, prior toinoculation with the cells of the invention, nylon matrices could betreated with 0.1 molar acetic acid and incubated in polylysine, PBS,and/or collagen to coat the nylon. Polystyrene could be similarlytreated using sulfuric acid.

In addition, the external surfaces of the three-dimensional frameworkmay be modified to improve the attachment or growth of cells anddifferentiation of tissue, such as by plasma coating the framework oraddition of one or more proteins (e.g., collagens, elastic fibers,reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, amongothers.

In some embodiments, the scaffold is comprised of or is treated withmaterials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as ePTFE, and segmented polyurethaneureasilicones, such as PURSPAN (The Polymer Technology Group, Inc.,Berkeley, Calif.). These materials can be further treated to render thescaffold non-thrombogenic. Such treatments include anti-thromboticagents such as heparin, and treatments which alter the surface charge ofthe material such as plasma coating.

Different proportions of the various types of collagen, for example,deposited on the framework can affect the growth of tissue-specific orother cells which may be later inoculated onto the framework or whichmay grow onto the structure in vivo. For example, for three-dimensionalskin culture systems, collagen types I and III are preferably depositedin the initial matrix. Alternatively, the framework can be inoculatedwith a mixture of cells which synthesize the appropriate collagen typesdesired. Thus, depending upon the tissue to be cultured, the appropriatecollagen type to be inoculated on the framework or produced by the cellsseeded thereon may be selected. For example, the relative amounts ofcollagenic and elastic fibers present in the framework can be modulatedby controlling the ratio of collagen-producing cells toelastin-producing cells in the initial inoculum. For example, since theinner walls of arteries are rich in elastin, an arterial scaffold shouldcontain a co-culture of smooth muscle cells which secrete elastin.

The seeded or inoculated three-dimensional framework of the inventioncan be used in a variety of applications. These include but are notlimited to transplantation or implantation of either the cultured cellsobtained from the matrix or the cultured matrix itself in vivo. Thethree-dimensional scaffolds may, according to the invention, be used toreplace or augment existing tissue, to introduce new or altered tissue,to modify artificial prostheses, or to join together biological tissuesor structures. For example, and not by way of limitation, specificembodiments of the invention include but are not limited to, flatstructures and tubular three-dimensional tissue implants for repair orregeneration, for example, of cardiac muscle, its structures, and thoseof the entire vascular tree, including for example the endovascularstructures of the brain and intracranium.

PPDCs can be inoculated onto a flat scaffold. The scaffold is preferablyincubated in culture medium prior to implantation. Two or more flatframeworks can be laid atop another and sutured together to generate amultilayer framework.

For example and not by way of limitation, the three-dimensionalframework can also be used to construct single and multi-layer tubulartissues in vitro that can serve as a replacement for damaged or diseasedtubular tissue in vivo.

A scaffold can be cut into a strip (e.g., rectangular in shape) of whichthe width is approximately equal to the inner circumference of thetubular organ into which it will ultimately be inserted. The cells canbe inoculated onto the scaffold and incubated by floating or suspendingin liquid media. At the appropriate stage of confluence, the scaffoldcan be rolled up into a tube by joining the long edges together. Theseam can be closed by suturing the two edges together using fibers of asuitable material of an appropriate diameter.

According to the invention, a scaffold can be formed as a tube,inoculated with PPDCs, and suspended in media in an incubation chamber.In order to prevent cells from occluding the lumen, one of the open endsof the tubular framework can be affixed to a nozzle. Liquid media can beforced through this nozzle from a source chamber connected to theincubation chamber to create a current through the interior of thetubular framework. The other open end can be affixed to an outflowaperture which leads into a collection chamber from which the media canbe recirculated through the source chamber. The tube can be detachedfrom the nozzle and outflow aperture when incubation is complete. Thismethod is described by Ballermann, B. J., et al., Int. Application No.WO 94/25584 and in U.S. application Ser. No. 08/430,768, both of whichare incorporated herein by reference in its entirety.

In general, two three-dimensional frameworks can be combined into a tubein accordance with the invention using any of the following methods.

Two or more flat frameworks can be laid atop another and suturedtogether. This two-layer sheet can then be rolled up, and, as describedabove, joined together and secured.

One tubular scaffold that is to serve as the inner layer can beinoculated with PPDCs and incubated. A second scaffold can be grown as aflat strip with width slightly larger than the outer circumference ofthe tubular framework. After appropriate growth is attained, the flatframework can be wrapped around the outside of the tubular scaffoldfollowed by closure of the seam of the two edges of the flat frameworkand, preferably, securing the flat framework to the inner tube.

Two or more tubular meshes of slightly differing diameters can be grownseparately. The framework with the smaller diameter can be insertedinside the larger one and secured.

For each of these methods, more layers can be added by reapplying themethod to the double-layered tube. The scaffolds can be combined at anystage of growth of the PPDCs, and incubation of the combined scaffoldscan be continued when desirable.

The lumenal aspect of the tubular construct can be comprised of ortreated with materials that render the lumenal surface of the tubularscaffold non-thrombogenic. These treatments and materials may alsopromote and sustain endothelial growth, migration, and extracellularmatrix deposition. Examples of these materials and treatments includebut are not limited to natural materials such as basement membraneproteins such as laminin and Type IV collagen, synthetic materials suchas ePTFE, and segmented polyurethaneurea silicones, such as PURSPAN (ThePolymer Technology Group, Inc., Berkeley, Calif.). These materials canbe further treated to render the lumenal surface of the tubular scaffoldnon-thrombogenic. Such treatments include anti-thrombotic agents such asheparin, and treatments which alter the surface charge of the materialsuch as plasma coating.

In conjuction with the above, the cells, cell lysates and fractions, andtherapeutic compositions of the invention can be used in conjunctionwith implantable devices. For example the cells, cell lysates and cellfractions can be coadminstered with, for example stents, artificialvalves, ventricular assist devices, Guglielmi detachable coils and thelike. As the devices may constitute the dominant therapy provided to anindividual in need of such therapy, the cells and the like may be usedas supportive or secondary therapy to assist in, stimulate, or promoteproper healing in the area of the implanted device. The cells, lysates,cell fractions and therapeutic compositions of the invention may also beused to “pretreat” certain implantable devices, to minimize problemswhen they are used in vivo. Such pretreated devices, including coateddevices may be better tolerated by patients receiving them, withdecrease risk of local or systemic infection, or for example, restenosisor further occulision of blood vessels.

The therapeutic cell compositions, in certain embodiments also comprisecells that express at least one of interleukin 8; reticulon 1; chemokine(C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha);chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2);chemokine (C-X-C motif) ligand 3; and tumor necrosis factor,alpha-induced protein 3; or which have reduced expression, relative to ahuman cell that is a fibroblast, a mesenchymal stem cell, or an ileaccrest bone marrow cell, for at least one of: short stature homeobox 2;heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeobox 2 (growth arrest-specifichomeobox); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; dishevelled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;B-cell translocation gene 1, anti-proliferative; cholesterol25-hydroxylase; runt-related transcription factor 3; hypotheticalprotein FLJ23191; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, alpha 7; DKFZP586L151 protein; transcriptionalco-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog2 (Drosophila); KIAA1034 protein; early growth response 3; distal-lesshomeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-likerepeat domains); Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic peptide receptor C);hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222(from clone DKFZp564B222); vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa.

Preferred therapeutic cell compositions also comprise cells whichsecrete at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF,BDNF, TPO, MIP1a, RANTES, and TIMP 1; and

-   -   do not secrete at least one of TGF-beta2, ANG2, PDGFbb, MIP1b,        I309, MDC, and VEGF, as detected by ELISA.

In yet another of its aspects, the invention provides methods fortreating a patient with a heart disease or injury comprisingadministering a therapeutic postpartum-derived cell composition to apatient with a disease or injury of the heart or circulatory system; andevaluating the patient for improvements in cardiac function. In certainpreferred embodiments the heart disease is a cardiomyopathy, eitheridiopathic or with a known cause, and either ischemic or nonischemic innature. While patients with any heart or circulatory disease willbenefit from such therapy, patients with myocardial infarction caused byany condition may benefit by receiving the therapeutic cell compositionsof the invention as discussed below. In other preferred embodiments, thedisease of the heart or circulatory system comprises one or more ofangioplasty, aneurysm, angina (angina pectoris), aortic stenosis,aortitis, arrhythmias, arteriosclerosis, arteritis, asymmetric septalhypertrophy (ASH), atherosclerosis, Athletic Heart Syndrome, atrialfibrillation and flutter, bacterial endocarditis, Barlow's Syndrome(mitral valve prolapse), bradycardia, Buerger's Disease (thromboangiitisobliterans), cardiomegaly, cardiomyopathy, carditis, carotid arterydisease, coarctation of the aorta, congenital heart diseases (congenitalheart defects), congestive heart failure (heart failure), coronaryartery disease, Eisenmenger's Syndrome, embolism, endocarditis,erythromelalgia, fibrillation, fibromuscular dysplasia, heart block,heart murmur, hypertension, hypotension, idiopathic infantile arterialcalcification, Kawasaki Disease (mucocutaneous lymph node syndrome,mucocutaneous lymph node disease, infantile polyarteritis), metabolicsyndrome, microvascular angina, myocardial infarction (heart attacks),myocarditis, paroxysmal atrial tachycardia (PAT), periarteritis nodosa(polyarteritis, polyarteritis nodosa), pericarditis, peripheral vasculardisease, phlebitis, pulmonary valve stenosis (pulmonic stenosis),Raynaud's Disease, renal artery stenosis, renovascular hypertension,rheumatic heart disease, septal defects, silent ischemia, syndrome X,tachycardia, Takayasu's Arteritis, Tetralogy of Fallot, transposition ofthe great vessels, tricuspid atresia, truncus arteriosus, valvular heartdisease, varicose ulcers, varicose veins, vasculitis, ventricular septaldefect, Wolff-Parkinson-White Syndrome, and endocardial cushion defect.

In still other preferred embodiments, the disease of the heart orcirculatory system comprises one or more of acute rheumatic fever, acuterheumatic pericarditis, acute rheumatic endocarditis, acute rheumaticmyocarditis, chronic rheumatic heart diseases, diseases of the mitralvalve, mitral stenosis, rheumatic mitral insufficiency, diseases ofaortic valve, diseases of other endocardial structures, ischemic heartdisease (acute and subacute), angina pectoris, diseases of pulmonarycirculation (acute pulmonary heart disease, pulmonary embolism, chronicpulmonary heart disease), kyphoscoliotic heart disease, pericarditis,myocarditis, endocarditis, endomyocardial fibrosis, endocardialfibroelastosis, atrioventricular block, cardiac dysrhythmias, myocardialdegeneration, diseases of the circulatory system includingcerebrovascular disease, occlusion and stenosis of precerebral arteries,occlusion of cerebral arteries, diseases of arteries, arterioles andcapillaries (atherosclerosis, aneurysm) diseases of veins andlymphatics, and other diseases of circulatory system.

Measurement of improvement in patients receiving the therapeuticcompositions provided herein can include any means known in the art, butpreferred improvements include improvements in hemodynamic measurementsincluding but not limited to chest cardiac output (CO), cardiac index(CI), pulmonary artery wedge pressures (PAWP), and cardiac index (CI), %fractional shortening (% FS), ejection fraction (EF), left ventricularejection fraction (LVEF); left ventricular end diastolic diameter(LVEDD), left ventricular end systolic diameter (LVESD), contractility(e.g. dP/dt), pressure-volume loops, measurements of cardiac work, anincrease in atrial or ventricular functioning; an increase in pumpingefficiency, a decrease in the rate of loss of pumping efficiency, adecrease in loss of hemodynamic functioning; and a decrease incomplications associated with cardiomyopathy. Biochemical measurementsof improvement are also contemplated herein, for example production ofcertain cellular products or factors. The presence or absence ofbiological molecules, for example particular enzymes (or theiractivities), mRNAs, transcription factors, proteins, modified proteins,lipids, sterols, or the like may be shown to correlate with improvementin cardiac or circulatory health and the use of these measurements ofimprovement are also contemplated for use herein. Also contemplatedherein as a indications of improvement are histological changes deemedbeneficial or for example, indicia of angiogenesis or improvedvascularization.

In some presently preferred embodiments, the methods comprise inducingthe therapeutic postpartum-derived cells to differentiate alongmesenchymal lineage, towards cardiomyogenic, angiogenic and vasculogenicphenotypes, or even further towards cells such as cardiomyocytes,endothelial cells, myocardial cells, epicardial cells, vascularendothelial cells, smooth muscle cells (e.g. vascular smooth musclecells), or towards cells of the excitatory and conductive systems, andprogenitors or more primitive relatives of the foregoing. Such cells arediscussed above, and the methods and factors for differentiating thecells, assessing the induction of cells to differentiate, and the usesof such cells for the therapeutic compositions is analogous. Also thetherapeutic cell compositions can integrate into the patient's heart, oralternatively can provide support for growth or stimulation todifferentiate for naturally present cardiac stem cells present.Therapeutic cells can be coadministered with cell lysates, or with otherallogeneic, syngeneic or autologous cells. The survival of the cellsdelivered in administering the therapeutic cell compositions is notdeterminative of the success or results of their use, rather theimprovement in cardiac or circulatory health is outcome determinative.Thus, the cells need not integrate and beat with the patient's heart, orinto blood vessels, but rather the indicia of improvements in cardiac orcirculatory health in the patient before and after treatment preferablyinclude at least one of objective measurements of cardiac or circulatoryhealth, and subjective assessment (including self-assessment) of thepatient's condition. A successful treatment could thus comprisetreatment of a patient with a cardiomyopathy with a therapeutic cellcomposition comprising the PPDCs, in the presence or absence of anothercell type. For example, and not by way of limitation, the PPDCspreferably at least partially integrate, multiply, or survive in thepatient. In other preferred embodiments, the patient experiencesbenefits from the therapy, for example from the ability of the PPDCs tosupport the growth of other cells, including stem cells or progenitorcells present in the heart, from the tissue ingrowth or vascularizationof the tissue, and from the presence of beneficial cellular factors,chemokines, cytokines and the like, but the cells do not integrate ormultiply in the patient. In another embodiment, the patient benefitsfrom the therapeutic treatment with the PPDCs, but the cells do notsurvive for a prolonged period in the patient. In one embodiment, thecells gradually decline in number, viability or biochemical activity, inother embodiments, the decline in cells may be preceded by a period ofactivity, for example growth, division, or biochemical activity. Inother embodiments, senescent, nonviable or even dead cells are able tohave a beneficial therapeutic effect.

The administering is preferably in vivo by transplanting, implanting,injecting, fusing, delivering via catheter, or providing as amatrix-cell complex, or any other means known in the art for providingcell therapy.

Patients with myocardial infarction caused by any condition may benefitby receiving the therapeutic cell compositions of the invention. Suchtreatment is preferably provided within a reasonable therapeutic windowafter the cardiac event. Presently, it is preferred that treatment withthe cells or compositions of the invention be initiated within 30 daysof the myocardial infarction. Treatment within 1-21 days is preferred.It is also comtemplated herein that beneficial effects of certainapplications, for example by intravenous injection where the cells hometo the damaged site, will allow treatment far more rapidly. For example,it is presently contemplated that treatment in close temporal relationwith the myocardial infarction or similar cardiac event may bebeneficial. In preferred embodiments, treatment with the therapeuticcell compositions is within twenty four hours of the cardiac event. Alsopreferred is treatment within 12, 8, or even four hours. More preferablytreatment is given with two hours. Treatment within one hour of theevent is more preferred, with treatment within 30 minutes, or even 15minutes most preferred. Also provided herein are kits for use in thetreatment of myocardial infarction. The kits provide the therapeuticcell composition which can be prepared in a pharmaceutically acceptableform, for example by mixing with a pharmaceutically acceptable carrier,and an applicator, along with instructions for use. Ideally the kit canbe used in the field, for example in a physician's office, or by anemergency care provider to be applied to a patient diagnosed as havinghad a myocardial infarction or similar cardiac event.

The invention also provides in another aspect, methods for treating apatient with a disease of the heart or circulatory system comprisingadministering a therapeutic postpartum-derived cell composition to apatient with a disease of the heart or circulatory system; andevaluating the patient for improvements in cardiac function, wherein theadministering is with a population of another cell type. Administrationof cocultures, mixed populations or other nonclonal populations arepreferred. Other cell types which can be coadministered are stem cellsin certain embodiments, while in others, myoblasts, myocytes,cardiomyoblasts, cardiomyocytes, or progenitors of myoblasts, myocytes,cardiomyoblasts, or cardiomyocytes are used.

Also provided herein are methods for treating a patient with a diseaseof the heart or circulatory system comprising administering atherapeutic postpartum-derived cell composition to a patient with adisease of the heart or circulatory system; and evaluating the patientfor improvements in cardiac function, wherein the therapeutic cellcomposition is administered as a matrix-cell complex. In certainembodiments, the matrix is a scaffold, preferably bioabsorbable,comprising at least the postpartum-derived cells.

Kits for the therapeutic application of the populations and coculturesof the invention are also provided. Where used for treatment ofcardiomyopathy, or other scheduled treatment, the kits include atherapeutic cell composition, with or without a matrix, and with orwithout a coculture present. The kits also optionally include a means ofadministering the cells, for example by injection, and apharmaceutically-acceptable carrier for the cells, if required. The kitsinclude instructions for use of the cells. Kits prepared for fieldhospital use, such as for military use may include full-proceduresupplies including tissue scaffolds, surgical sutures, and the like,where the cells are to be used in conjunction with repair of acutecardiac injuries.

The invention also provides for banking of tissues, cells, populationsand therapeutic cell compositions of the invention. As discussed abovethe cells are readily cryopreserved. The invention therefore providesmethods of cryopreserving the cells in a bank, wherein the cells arestored frozen and associated with a complete characterization of thecells based on immunological, biochemical and genetic properties of thecells. The cells so frozen can be used for autologous, syngeneic, orallogeneic therapy, depending on the requirements of the procedure andthe needs of the patient. Preferably, the information on eachcryopreserved sample is stored in a computer which is searchable basedon the requirements of the surgeon, procedure and patient with suitablematches being made based on the characterization of the cells orpopulations. Preferably, the cells of the invention are grown andexpanded to the desired quantity of cells and therapeutic cellcompositions are prepared either separately or as cocultures, in thepresence or absence of a matrix or support. While for some applicationsit may be preferable to use cells freshly prepared, the remainder can becryopreserved and banked by freezing the cells and entering theinformation in the computer to associate the computer entry with thesamples. Even where it is not necessary to match a source or donor witha recipient of such cells, for immunological purposes, the bank systemmakes it easy to match, for example, desirable biochemical or geneticproperties of the banked cells to the therapeutic needs. Upon matchingof the desired properties with a particle banked sample, the sample isretrieved and readied for therapeutic use. Cell lysates prepared asdescribed herein may also be cryopreserved and banked in accordance withthe present invention.

In another aspect of the invention, kits for the growth and maintenance,the isolation and the use of the umbilical-derived cells are provided.The cells, cell lysates, soluble cell fractions, membrane fractions andmatrices can conveniently be employed as parts of kits, for example, fora kit for culture or implantation. The invention provides a kitincluding the UDCs and additional components, including instructions forgrowth or maintenance, isolation, or use of the cells or cell fractions,together with for example, matrix (e.g., a scaffold) material, hydratingagents (e.g., physiologically-compatible saline solutions, prepared cellculture media), cell culture substrates (e.g., culture dishes, plates,vials, etc.), cell culture media (whether in liquid or dehydrated form),antibiotic compounds, hormones, and the like. Kits for growth can forexample include all of the components of the Growth Medium as usedherein, including serum, for example fetal bovine serum. While the kitcan include any such components, preferably it includes all ingredientsnecessary for its intended use. If desired, the kit also can includecells (typically cryopreserved), which can be seeded into the lattice asdescribed herein. Kits for isolation will contain everything required topractice the isolation methods as provided herein, except for theumbilicus tissue which should be obtained fresh or frozen from a tissuebank at the time of isolation. The surgical equipment for dissociatingthe tissue, preferred enzymes, or choices of enzymes in stable form areprovided, as are the buffers and medium, cell strainers and the like, asrequired or preferred for the method as disclosed above. Detailedinstructions with optional steps and lists of suppliers of optional oralternative materials are alos conveniently provided. Control cells canbe included for comparison of the cells isolated to, for example the UDCcultures deposited with the ATCC. Kits for utilizing theumbilicus-derived cells preferably contain populations of the cells, ortherapeutic compositions comprising the cells, components and products,or fractions or conditioned media derived from the cells as describedabove. In some embodiments, the kits may include one or more cellpopulations, including at least UDCs and a pharmaceutically acceptablecarrier (liquid, semi-solid or solid). The populations in someembodiments are homogenous or even clonal cell lines of UDCs. In otherembodiments, the kits include other cell lines for use in coculture.Therapeutic application kits preferably include additional bioactiveagents as desired for example anithrombogenic agents, anti-inflammatoryagents, antiapoptotic agents, and immunosuppressive or immunomodulatorycompounds. The kits also optionally may include a means of administeringthe cells, for example by injection. The kits further may includeinstructions for use of the cells. Kits prepared for field hospital use,such as for military use, may include full-procedure supplies includingtissue scaffolds, surgical sutures, and the like, where the cells are tobe used in conjunction with repair of acute injuries. Kits for assaysand in vitro methods as described herein may contain one or more of (1)UDCs or fractions, components or products of UDCs, (2) reagents forpracticing the in vitro method, (3) other cells or cell populations, asappropriate, for example for cocultures and (4) instructions forconducting the in vitro method. Kits for the preparation of cell-derivedcomponenets can include both the components required for growth of thecells and the components required for preparing the cell fraction ofinterest, along with instructions for obtaining the desried fractionfrom the cells. Kits for production of and collection of conditionedmedia are also provided herein and include cells, medium, collectionvessels, instructions, standards for assaying the secreted molecules ofinterest and the like.

The following examples describe several aspects of embodiments of theinvention in greater detail. These examples are provided to furtherillustrate, not to limit, aspects of the invention described herein.

EXAMPLE 1 Isolation of Cells from Postpartum Tissues

Summary

Postpartum cells have been isolated from full- and pre-term placentaland umbilical cord tissues. A highly preferred way of isolating cellsfrom these tissues is by using a combination of digestive enzymes.Particularly preferred are collagenase, hyaluronidase and dispase. Thiscombination results in the isolation of a cell population with goodexpansion and differentiation potentials. Other enzyme combinations usedhave yielded cell populations that can also be expanded.

Introduction

Populations of cells from placental and umbilical cord tissues wereisolated. Postpartum umbilicus and placenta were obtained upon births ofeither full- or pre-term pregnancies. Cells were harvested from fiveseparate donors of umbilicus and placental tissue. Different methods ofcell isolation were tested for their ability to yield cells with: 1) thepotential to differentiate into cells with different phenotypes, acharacteristic common to stem cells, or 2) the potential to providecritical trophic factors useful for other cells and tissues.

Methods & Materials

Umbilical Cell Isolation

Umbilical cords were obtained from National Disease Research Interchange(NDRI, Philadelphia, Pa.). The tissues were obtained following normaldeliveries. The cell isolation protocol was performed aseptically in alaminar flow hood. To remove blood and debris, the cord was washed inphosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in thepresence of 10,000 Units of antimycotic and antibiotic per 100milliliters of PBS (Invitrogen Carlsbad, Calif.). The tissues were thenmechanically dissociated in 150 cm² tissue culture plates in thepresence of 50 milliliters of medium (DMEM-Low glucose or DMEM-Highglucose; Invitrogen), until the tissue was minced into a fine pulp. Thechopped tissues were transferred to 50 milliliter conical tubes(approximately 5 grams of tissue per tube). The tissue was then digestedin either DMEM-Low glucose medium or DMEM-High glucose medium, eachcontaining 10,000 Units of antimycotic and antibiotic per 100milliliters of PBS and digestion enzymes. In some experiments an enzymemixture of collagenase and dispase was used (“C:D;” collagenase (Sigma,St Louis, Mo.), 500 Units/milliliter; and dispase (Invitrogen), 50Units/milliliter in DMEM:-Low glucose medium). In other experiments amixture of collagenase, dispase and hyaluronidase (“C:D:H”) was used(collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter; andhyaluronidase (Sigma), 5 Units/milliliter, in DMEM:-Low glucose). Theconical tubes containing the tissue, medium and digestion enzymes wereincubated at 37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth Medium (DMEM:Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 1milliliter per 100 milliliters of antibiotic/antimycotic (10,000 Unitsper milliliter penicillin, 10,000 micrograms per milliliterstreptomycin, 25 micrograms per milliliter amphotericin B; Invitrogen,Carlsbad, Calif.)). The cell suspension was filtered through a70-micrometer nylon cell strainer (Nalge Nunc International, Rochester,N.Y.). An additional 5 milliliters rinse comprising Growth Medium waspassed through the strainer. The cell suspension was then passed througha 40-micrometer nylon cell strainer (Nalge Nunc International) andchased with a rinse of an additional 5 milliliters of Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh Growth Medium. Thenumber of viable cells was determined using trypan blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cord cells were seeded at 5,000cells/cm² onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning,N.Y.) in Growth Medium. After 2 days, spent medium was aspirated fromthe flasks. Cells were washed with PBS three times to remove debris andblood-derived cells. Cells were then replenished with Growth Medium andallowed to grow to confluence (about 10 days from passage 0) topassage 1. On subsequent passages (from passage 1 to 2 etc), cellsreached sub-confluence (75-85 percent confluence) in 4-5 days. For thesesubsequent passages, cells were seeded at 5000 cells/cm². Cells weregrown in a humidified incubator with 5 percent carbon dioxide and 20percent oxygen, at 37° C.

Placental Cell Isolation

Placental tissue was obtained from NDRI (Philadelphia, Pa.). The tissueswere from a pregnancy and were obtained at the time of a normal surgicaldelivery. Placental cells were isolated as described for umbilical cellisolation.

The following description applies to the isolation of separatepopulations of maternal-derived and neonatal-derived cells fromplacental tissue.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (penicillin, 10,000 Units/milliliter; streptomycin, 10,000micrograms/milliliter; amphotericin B, 25 micrograms/milliliter;Invitrogen) to remove as much blood and debris as practical. Theplacental tissue was then dissected into three sections: neonatalaspect, the villous region, the maternal aspect.

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. In thismanner, substantially all the blood was removed. Each section was thenmechanically dissociated in 150 cm² tissue culture plates in thepresence of 50 milliliters of DMEM:Low glucose (Invitrogen), to a finepulp. The pulp was transferred to 50 milliliter conical tubes. Each tubecontained approximately 5 grams of tissue. The tissue was digested ineither DMEM-Low glucose DMEM-High glucose medium containing 10,000 Unitsof antimycotic and antibiotic per 100 milliliters of PBS and digestionenzymes. In some experiments an enzyme mixture of collagenase anddispase (“C:D”) was used containing collagenase (Sigma, St Louis, Mo.)at 500 Units/milliliter and dispase (Invitrogen) at 50 Units/milliliterin DMEM:-Low glucose medium. In other experiments, a mixture ofcollagenase, dispase and hyaluronidase (C:D:H) was used (collagenase,500 Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase(Sigma), 5 Units/milliliter in DMEM:-Low glucose). The conical tubescontaining the tissue, medium, and digestion enzymes were incubated for2 h at 37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the resultant supernatant was aspirated off. The pellet was resuspendedin 20 milliliters of Growth Medium. The cell suspension was filteredthrough a 70 micrometer nylon cell strainer (Nalge Nunc International,Rochester, N.Y.), and rinsed with 5 milliliters of Growth Medium. Thetotal cell suspension was passed through a 40 micrometer nylon cellstrainer (Nalge Nunc International) and rinsed with an additional 5milliliters of Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cell pellet was resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more. After the finalcentrifugation, supernatant was aspirated and the cell pellet wasresuspended in 5 milliliters of fresh Growth Medium. A cell count wasdetermined using the trypan blue Exclusion test. Cells were culturedunder standard conditions.

LIBERASE Cell Isolation

Cells were isolated from postpartum tissues in DMEM-Low glucose mediumwith LIBERASE (2.5 milligrams per milliliter, Blendzyme 3; (RocheApplied Sciences, Indianapolis, Ind.)) and hyaluronidase (5Units/milliliter, Sigma). Digestion of the tissue and isolation of thecells was as described for other digestions above, with theLIBERASE/hyaluronidase mixture used instead of the C:D or C:D:H enzymemixture. Tissue digestion with LIBERASE resulted in the isolation ofcell populations from postpartum tissues that expanded readily.

Cell Isolation Using Other Enzyme Combinations

Procedures were compared for isolating cells from the umbilical cordusing differing enzyme combinations. Enzymes compared for digestionincluded: i) collagenase; ii) dispase; iii) hyaluronidase; iv)collagenase:dispase mixture (C:D); v) collagenase:hyaluronidase mixture(C:H); vi) dispase:hyaluronidase mixture (D:H); and vii)collagenase:dispase:hyaluronidase mixture (C:D:H). Differences in cellisolation utilizing these different enzyme digestion conditions wereobserved (Table 1-1).

Isolation of Cells from Residual Blood in the Cords

Other attempts were made to isolate pools of cells from umbilical cordsby different approaches. In one instance umbilical cord was sliced andwashed with Growth Medium to dislodge the blood clots and gelatinousmaterial. The mixture of blood, gelatinous material and Growth Mediumwas collected and centrifuged at 150×g. The pellet was resuspended andseeded onto gelatin coated flasks in Growth Medium. Cell populationsthat readily expanded were isolated.

Isolation of Cells from Cord Blood

Cells were also isolated from cord blood samples attained from NDRI. Theisolation protocol used here was that of International PatentApplication US0229971 by Ho et al. Samples (50 milliliter and 10.5milliliters, respectively) of umbilical cord blood (NDRI, PhiladelphiaPa.) were mixed with lysis buffer (filter-sterilized 155 millimolarammonium chloride, 10 millimolar potassium bicarbonate, 0.1 millimolarEDTA buffered to pH 7.2 (all components from Sigma, St. Louis, Mo.)).Cells were lysed at a ratio of 1:20 cord blood to lysis buffer. Theresulting cell suspension was vortexed for 5 seconds, and incubated for2 minutes at ambient temperature. The lysate was centrifuged (10 minutesat 200×g). The cell pellet was resuspended in complete minimal essentialmedium (Gibco, Carlsbad Calif.) containing 10 percent fetal bovine serum(Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech Herndon, VA),100 Units penicillin per 100 milliliters and 100 micrograms streptomycinper 100 milliliters (Gibco, Carlsbad, Calif.). The resuspended cellswere centrifuged (10 minutes at 200×g), the supernatant was aspirated,and the cell pellet was washed in complete medium. Cells were seededdirectly into either T75 flasks (Corning, N.Y.), T75 laminin-coatedflasks, or T175 fibronectin-coated flasks (both Becton Dickinson,Bedford, Mass.).

Isolation of Postpartum Cells Using Different Enzyme Combinations andGrowth Conditions

To determine whether cell populations can be isolated under differentconditions and expanded under a variety of conditions immediately afterisolation, cells were digested in Growth Medium with or without 0.001percent (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using the C:D:Henzyme combination, according to the procedures provided above.Placental cells so isolated were seeded under a variety of conditions.All cells were grown in the presence of penicillin/streptomycin. (Table1-2).

Isolation of Postpartum Cells Using Different Enzyme Combinations andGrowth Conditions

In all conditions tested, cells attached and expanded well from aboutpassage 0 to 1 (Table 1-2). Cells in conditions 5-8 and 13-16proliferated well up to 4 passages after seeding at which point theywere cryopreserved. All cells were banked for later investigation.

Results

Cell Isolation Using Different Enzyme Combinations

The combination of C:D:H enzymes provided the best cell yield followingisolation, and generated cells which expanded for many more generationsin culture than the other conditions (Table 1). An expandable cellpopulation was not attained using collagenase or hyaluronidase alone. Noattempt was made to determine if this result is specific to thecollagenase that was tested.

Isolation of Postpartum Cells Using Different Enzyme Combinations andGrowth Conditions

Cells attached and expanded well from about passage 0 to 1 under allconditions tested for enzyme digestion and growth (Table 1-2). Cells inexperimental conditions 5-8 and 13-16 proliferated well up to 4 passagesafter seeding, at which point they were cryopreserved. All cells werebanked for further investigation.

Isolation of Cells from Residual Blood in the Cords

Nucleated cells attached and grew rapidly. These cells were analyzed byflow cytometry and were similar to cells obtained by enzyme digestion.

Isolation of Cells from Cord Blood

The preparations contained red blood cells and platelets. No nucleatedcells attached and divided during the first 3 weeks. The medium waschanged 3 weeks after seeding and no cells were observed to attach andgrow.

DISCUSSION AND CONCLUSION

Populations of cells can be isolated from umbilical and placental tissuemost efficiently using the enzyme combination collagenase (ametalloprotease), dispase (a neutral protease) and hyaluronidase (amucolytic enzyme which breaks down hyaluronic acid). LIBERASE Blendzyme,which is a commercial blend of collagenase and another protease may alsobe used. In the present study Blendzyme 3 which contains collagenase (4Wunsch units/g) and thermolysin (1714 casein Units/g) was also usedtogether with hyaluronidase to isolate cells. Cells isolated with theseenzymes expand readily over many passages, for example, when cultured inGrowth Medium on gelatin coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue, thatadhere and grow under the conditions used, may be due to cells beingreleased during the dissection process. Other explanations may includethe migration of cells from the matrix.

Recommendation

Use of C:D:H enzyme combinations for isolation of cell populations frompostpartum tissues is preferred. Cells isolated using this combinationof enzymes have been extensively characterized and have many desirableproperties. LIBERASE extracted cells and cells treated with other enzymecombination cells provide useful cells with expansion potential. It maybe useful to choose a process or method for cell isolation that helpsminimize handling and transfer of the tissue. Such methods may includemechanical digestion for example with a blender, tissue homogenizer, andthe like.

REFERENCE

s1. H O, Tony, W.; KOPEN, Gene, C.; RIGHTER, William, F.; RUTKOWSKI, J.,Lynn; HERRING, W., Joseph; RAGAGLIA, Vanessa; WAGNER, JosephWO2003025149 A2 CELL POPULATIONS WHICH CO-EXPRESS CD49C AND CD90,NEURONYX, INC. Application No. US0229971 US, Filed 20020920, A2Published 20030327, A3 Published 20031218 TABLE 1-1 Isolation of cellsfrom umbilical cord tissue using varying enzyme combinations EnzymeDigest Cells Isolated Cell Expansion Collagenase X X Dispase + (>10 h) +Hyaluronidase X X Collagenase:Dispase ++ (<3 h) ++Collagenase:Hyaluronidase ++ (<3 h) + Dispase:Hyaluronidase + (>10 h) +Collagenase:Dispase:Hyaluronidase +++ (<3 h) +++Key:+ = good,++ = very good,+++ = excellent,X = no success

TABLE 1-2 Isolation and culture expansion of postpartum cells undervarying conditions: 15% 20% Growth Condition Medium FBS BME Gelatin O₂Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N N (5%) 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N N (5%) 5 DMEM-Lg N Y N Y EGF/FGF (2%) (Laminin) (20ng/ milliliter) 6 DMEM-Lg N Y N N EGF/FGF (2%) (Laminin) (5%) (20 ng/milliliter) 7 DMEM-Lg N Y N Y PDGF/ (2%) (Fibrone) VEGF 8 DMEM-Lg N Y NN PDGF/ (2%) (Fibrone) (5%) VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y NN (5%) 11 DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N N (5%) 13 DMEM-Lg N N N YEGF/FGF (2%) (Laminin) (20 ng/ milliliter) 14 DMEM-Lg N N N N EGF/FGF(2%) (Laminin) (5%) (20 ng/ milliliter) 15 DMEM-Lg N N N Y PDGF/ (2%)(Fibrone) VEGF 16 DMEM-Lg N N N N PDGF/ (2%) (Fibrone) (5%) VEGF

EXAMPLE 2 Growth Characteristics of Postpartum Cells

Summary

Commercially viable cell products must be able to be produced insufficient quantities to provide therapeutic treatment to patients inneed of the treatment. Postpartum cells can be expanded in culture forsuch purposes. Comparisons were made of the growth of postpartum cellsin culture to that of other cell populations including mesenchymal stemcells. The data demonstrate that postpartum cell lines as developedherein can expand for greater than 40 doublings to provide sufficientcell numbers, for example, for pre-clinical banks. Furthermore, thesepostpartum cell populations can be expanded well from low- orhigh-density seeding. This study has demonstrated that mesenchymal stemcells, in contrast, cannot be expanded to obtain large quantities ofcells.

Introduction

The cell expansion potential of postpartum cells was compared to otherpopulations of isolated stem cells. The art of cell expansion tosenescence is referred to as Hayflick's limit (Hayflick L. The longevityof cultured human cells. J. Am. Geriatr. Soc. 22(1):1-12, 1974; HayflickL. The strategy of senescence. Gerontologist 14(1):37-45), 1974).Senescence is defined as the point at which cell division stopscompletely, i.e., when the cell loses its ability to proliferate andexpand. Postpartum-derived cells are highly suited for therapeutic usebecause they can be readily expanded to sufficient cell numbers.

Materials and Methods

Gelatin-Coating Flasks

Tissue culture plastic flasks were coated by adding 20 milliliters of a2% (w/v) gelatin (Type B: 225 Bloom; Sigma, St Louis, Mo.) solution eachto T75 flasks (Corning, Corning, N.Y.) for 20 minutes at roomtemperature. After removing the gelatin solution, 10 millilitersphosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) wereadded and then aspirated.

Comparison of Expansion Potential of Postpartum Cells vs. Other StemCell and Non-Stem Cell Populations

For comparison of growth expansion potential the following cellpopulations were utilized; i) Mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) Adipose-derived cells (US6555374 B1; U.S. PatentApplication US20040058412); iii) Normal dermal skin fibroblasts (cc-2509lot # 9F0844; Cambrex, Walkersville, Md.); iv) Umbilical-derived cells;and vi) Placental-derived cells. Cells were initially seeded at 5,000cells/cm² on gelatin-coated T75 flasks in Growth Medium.

For subsequent passages, cell cultures were treated as follows: Aftertrypsinization, viable cells were counted after trypan blue staining.Cell suspension (50 microliters) was combined with trypan blue (50 ml,Sigma, St. Louis Mo.). Viable cell numbers were estimated using ahemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 ml of fresh Growth Medium. Cells weregrown under standard atmospheric conditions (5 percent carbon dioxide)at 37° C. in the presence of 20 percent oxygen and 75 percent nitrogen(v/v). The Growth Medium was changed twice per week. When cells reachedabout 85 percent confluence they were passaged; this process wasrepeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [ln (cell final/cell initial)/ln2] anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.expansion factor=cell final/cell initial).

Expansion of Potential of Cell Banks at Low Density

The expansion potential of cells banked at passage 10 was also tested. Adifferent set of conditions was used. Normal dermal skin fibroblasts(cc-2509 lot # 9F0844; Cambrex, Walkersville, Md.), umbilical-derivedcells, and placenta-derived cells were tested. These cell populationshad been banked at passage 10 previously, having been cultured at 5,000cells/cm² at each passage to that point. The effect of cell density onthe cell populations following cell thaw at passage 10 was determined.Cells were thawed under standard conditions, counted using trypan bluestaining. Thawed cells were then seeded at 1000 cells/cm² in GrowthMedium. Cells were grown under standard atmospheric conditions at 37° C.Growth Medium was changed twice a week and cells were passaged as theyreached about 85% confluence. Cells were subsequently passaged untilsenescence, i.e., until they could not be expanded any further. Cellswere trypsinized and counted at each passage. The cell yield, populationdoubling (ln (cell final/cell initial)/ln2) and doubling time (time inculture (h)/population doubling). The total cell yield per passage wasdetermined by multiplying total yield for the previous passage by theexpansion factor for each passage (i.e., expansion factor=cellfinal/cell initial).

Expansion of Postpartum Cells at Low Density from Initial Cell Seeding

The expansion potential of freshly isolated postpartum cell culturesunder low cell seeding conditions was tested in another experiment.Umbilical and placental cells were isolated as described herein. Cellswere seeded at 1000 cells/cm² and passaged as described above untilsenescence. Cells were grown under standard atmospheric conditions at37° C. Growth Medium was changed twice per week. Cells were passaged asthey reached about 85% confluence. At each passage, cells weretrypsinized and counted by trypan blue staining. The cell yield,population doubling (In (cell final/cell initial)/ln2) and doubling time(time in culture (h)/population doubling) were calculated for eachpassage. The total cell yield per passage was determined by multiplyingthe total yield for the previous passage by the expansion factor foreach passage (i.e. expansion factor=cell final/cell initial). Cells weregrown on gelatin and non-gelatin coated flasks.

Expansion of Clonal Neonatal Placental Cells

Cloning was used in order to expand a population of neonatal cellssuccessfully from placental tissue. Following isolation of threedifferential cell populations from the placenta (as described herein),these cell populations were expanded under standard growth conditionsand then karyotyped to reveal the identity of the isolated cellpopulations. Since these cells were isolated from a mother who delivereda boy it was very simple to distinguish between the male and femalechromosomes by performing metaphase spreads. These experimentsdemonstrated that cells isolated from the neonatal aspect were primarilykaryotype-positive for neonatal phenotpye, and cells isolated from thematernal aspect were primarily karyotype-positive for maternal cells,those cells isolated from the villous region were karyotype-positive forboth neonatal and maternal phenotypes. Subcloning of populations derivedfrom neonatal and maternal aspects is required to ensure that clonalpopulations are obtained for both neonatal and maternal cells.

Expansion of Cells in Low Oxygen Culture Conditions

It has been demonstrated that low O₂ cell culture conditions can improvecell expansion in certain circumstances (Csete, Marie; Doyle, John;Wold, Barbara J.; McKay, Ron; Studer, Lorenz. Low oxygen culturing ofcentral nervous system progenitor cells. US20040005704). In order todetermine if cell expansion of postpartum-derived cells could beimproved by altering cell culture conditions, cultures ofumbilical-derived cells were grown in low oxygen conditions. Cells wereseeded at 5000 cells/cm² in Growth Medium on gelatin coated flasks.Cells were initially cultured under standard atmospheric conditionsthrough passage 5, at which point they were transferred to low oxygen(5% O₂) culture conditions.

Other Growth Conditions

In other experiments cells were expanded on non-coated, collagen-coated,fibronectin-coated, laminin-coated and Matrigel-coated plates. Cultureshave been demonstrated to expand well on these different matrices.

Results

Comparison of Expansion Potential of Postpartum Cells vs. Other StemCell and Non-Stem Cell Populations

Both umbilical-derived and placenta-derived cells expanded for greaterthan 40 passages generating cell yields of >1E17 cells in 60 days. Incontrast, MSCs and fibroblasts senesced after <25 days and <60 days,respectively. Although both adipose-derived and omental cells expandedfor almost 60 days they generated total cell yields of 4.5E12 and4.24E13 respectively. Thus, when seeded at 5000 cells/cm² under theexperimental conditions utilized, postpartum-derived cells expanded muchbetter than the other cell types grown under the same conditions (Table1).

Expansion of Potential of Cell Banks at Low Density

Umbilical-derived, placental-derived and fibroblast cells expanded forgreater than 10 passages generating cell yields of >1E11 cells in 60days (Table 2). After 60 days under these conditions the fibroblastsbecame senescent whereas the umbilical-derived and placental-derivedcell populations senesced after 80 days, completing >50 and >40population doublings respectively.

Expansion of Postpartum Cells at Low Density from Initial Cell Seeding

Placental-derived cells were expanded at low density (1,000 cells/cm²)on gelatin-coated and uncoated plates or flasks. Growth potential ofthese cells under these conditions was good. The cells expanded readilyin a log phase growth. The rate of cell expansion was similar to thatobserved when placental-derived cells were seeded at 5000 cells/cm² ongelatin-coated flasks in Growth Medium. No differences were observed incell expansion potential between culturing on either uncoated flasks orgelatin-coated flasks. However, cells appeared phenotypically muchsmaller on gelatin-coated flasks and more larger cell phenotypes wereobserved on uncoated flasks.

Expansion of Clonal Neonatal and Maternal Placental Cells

The expansion of a clonal cell populations of placental-derived cellsisolated from the neonatal and maternal aspects of the placenta arestudied. Populations derived from neotal and maternal aspects areserially diluted and then seeded onto gelatin-coated plates in GrowthMedium for expansion at 1 cell/well in 96-well gelatin coated plates.From this initial cloning, expansive clones are identified, trypsinizedand reseeded in 12 well gelatin coated plates in Growth Medium and thensubsequently passaged into T25 gelatin coated flasks at 5,000 cells/cm²in Growth Medium. Subcloning is then performed to ensure that a clonalpopulation of cells had been identified. For subcloning experimentscells are trypsinized and reseeded at 0.5 cells/well. The subclones thatgrow well are then expanded in gelatin-coated T25 flasks at 5,000cells/cm². Cells are subsequently passaged at 5,000 cells/cm² in T75flasks. Karyotyping confirms that clones so-derived are neotal ormaternal in nature.

Expansion of Cells in Low Oxygen Culture Conditions

Cells expanded well under the reduced oxygen conditions, however,culturing under low oxygen conditions does not appear to have asignificant effect on cell expansion for postpartum-derived cells. Theseresults are preliminary in the sense that any ultimate conclusions to bemade regarding the effect of reduced oxygen should include data fromexperiments on growing cells in low oxygen from initial isolation.Standard atmospheric conditions have already proven successful forgrowing sufficient numbers of cells, and low oxygen culture iscompatible with, but not required for, the growth of postpartum-derivedcells.

DISCUSSION AND CONCLUSIONS

The current cell expansion conditions of growing isolatedpostpartum-derived cells at densities of about 5000 cells/cm², in GrowthMedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggest that the cells can be readily expandedusing lower density culture conditions (e.g. 1000 cells/cm²).Postpartum-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing postpartum-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,postpartum-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, postpartum-derived cells expandreadily to large numbers.

Recommendations:

In order to optimize expansion and scale-up of postpartum-derived cellcultures additional work with new methods for cell expansion, mediaconditions, extracellular matrix conditions for cellular attachment andcell density would be useful. However, with all these changes the cellpotential would have to be re-determined.

REFERENCES

-   1) Hayflick L. The longevity of cultured human cells. J Am Geriatr    Soc. 1974 January; 22(1):1-12.-   2) Hayflick L. The strategy of senescence. Gerontologist. 1974    February;14(1):37-45.-   3) Patent US20040058412-   4) Patent US20040048372

6) Csete, Marie; (Ann Arbor, Mich.); Doyle, John; (South Pasadena,Calif.); Wold, Barbara J.; (San Marino, Calif.); McKay, Ron; (Bethesda,Md.); Studer, Lorenz; (New York, N.Y.). Low oxygen culturing of centralnervous system progenitor cells. US20040005704. TABLE 1 Growthcharacteristics for different cell populations grown to senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield MSc 24 d 84.72 E7 Adipose-derived 57 d 24  4.5 E12 Fibroblasts 53 d 26 2.82 E13Umbilical 65 d 42 6.15 E17 Placenta 80 d 46 2.49 E19

TABLE 2 Growth characteristics for different cell populations using lowdensity growth expansion from passage 10 till senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 d 43.68 2.59 E11 Umbilical (P10) 80 d 53.6 1.25 E14 Placental(P10) 60 d 32.96 6.09 E12

EXAMPLE 3 Growth of Postpartum Cells in Medium Containing D-Valine

Summary

Culture media containing D-valine instead of the L-valine isoformreportedly selectively inhibit the growth of fibroblast-like cells inculture. To determine whether postpartum-derived cells can grow inmedium containing D-valine, cells derived from placenta and umbilicalcord were grown in medium containing D-valine for 4 weeks. The cells didnot proliferate and eventually died. Medium containing D-valine is notsuitable for selectively growing postpartum-derived cells. L-valine isrequired for postpartum-derived cell proliferation and long-termviability.

Introduction

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). It was not previously known whether postpartum-derived cells cangrow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9) and umbilical-derivedcells (P5) were seeded at 5×10³ cells/cm² in gelatin-coated T75 flasks(Corning, Corning, N.Y.). After 24 hours the medium was removed and thecells were washed with phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) to remove residual medium. The medium was replaced with aModified Growth Medium (DMEM with D-valine (special order Gibco), 15%(v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma), penicillin/streptomycin (Gibco)).

Results

Placenta-derived, umbilical-derived, and fibroblast cells seeded in theD-valine-containing medium did not proliferate, unlike cells seeded inGrowth Medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after 4 weeks.

DISCUSSION AND CONCLUSION

Postpartum-derived cells require L-valine for cell growth and tomaintain long-term viability. L-valine should therefore not be removedfrom the Growth Medium for postpartum-derived cells.

REFERENCES

-   Hongpaisan J. (2000) Inhibition of proliferation of contaminating    fibroblasts by D-valine in cultures of smooth muscle cells from    human myometrium. Cell Biol Int. 24:1-7.

Sordillo L M, Oliver S P, Akers R M. (1988) Culture of bovine mammaryepithelial cells in D-valine modified medium: selective removal ofcontaminating fibroblasts. Cell Biol Int Rep. 12:355-64.

EXAMPLE 4 Karyotype Analysis of PPDCs

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Cells used in cell therapy should have anormal chromosome number (46) and structure. To identify postpartumplacental and umbilical cord cell lines that are homogeneous and freefrom cells of non-postpartum tissue origin, karyotypes of cell sampleswere analyzed.

Materials and Methods

PPDCs from postpartum tissue of a male neonate were cultured in GrowthMedia. Postpartum tissue from a male neonate (X,Y) was selected to allowdistinction between neonatal-derived cells and maternal derived cells(X,X). Cells were seeded at 5,000 cells per square centimeter in GrowthMedium in a T25 flask (Corning, Corning, N.Y.) and expanded to 80%confluence. A T25 flask containing cells was filled to the neck withGrowth Medium. Samples were delivered to a clinical cytogenetics lab bycourier (estimated lab to lab transport time is one hour). Chromosomeanalysis was performed by the Center for Human & Molecular) Genetics atthe New Jersey Medical School, Newark, N.J. Cells were analyzed duringmetaphase when the chromosomes are best visualized. Of twenty cells inmetaphase counted, five were analyzed for normal homogeneous karyotypenumber (two). A cell sample was characterized as homogeneous if twokaryotypes were observed. A cell sample was characterized asheterogeneous if more than two karyotypes were observed. Additionalmetaphase cells were counted and analyzed when a heterogeneous karyotypenumber (four) was identified.

Results

All cell samples sent for chromosome analysis were interpreted by thecytogenetics laboratory staff as exhibiting a normal appearance. Threeof the sixteen cell lines analyzed exhibited a heterogeneous phenotype(XX and XY) indicating the presence of cells derived from both neonataland maternal origins (Table 4-1). Cells derived from tissue Placenta-Nwere isolated from the neonatal aspect of placenta. At passage zero,this cell line appeared homogeneous XY. However, at passage nine, thecell line was heterogeneous (XX/XY), indicating a previously undetectedpresence of cells of maternal origin. TABLE 4-1 Karyotype results ofPPDCs. number Tissue passage Metaphase cells counted Metaphase cellsanalyzed of karyotype ISCN Karyotype Placenta 22 20 5 2 46, XX Umbilical23 20 5 2 46, XX Umbilical 6 20 5 2 46, XY Placenta 2 20 5 2 46, XXUmbilical 3 20 5 2 46, XX Placenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 246, XY Placenta-M 0 21 5 4 46, XY[18]/46, XX[3] Placenta-M 4 20 5 2 46,XX Placenta-N 9 25 5 4 46, XY[5]/46, XX[20] Placenta-N C1 1 20 5 2 46,XY Placenta-N C3 1 20 6 4 46, XY[2]/46, XX[18] Placenta-N C4 1 20 5 246, XY Placenta-N C15 1 20 5 2 46, XY Placenta-N C20 1 20 5 2 46, XYKey: N—Neonatal aspect; V—villous region; M—maternal aspect; C—clone

Summary

Chromosome analysis identified placenta- and umbilical cord-derivedPPDCs whose karyotypes appear normal as interpreted by a clinicalcytogenetic laboratory. Karyotype analysis also identified cell linesfree from maternal cells, as determined by homogeneous karyotype.

EXAMPLE 5 Evaluation of Postpartum-Derived Cell Surface Markers by FlowCytometry

Summary

Characterization of cell surface protein expression, or “markers” byflow cytometry of cultured cell lines labeled with fluorescentmonoclonal antibodies enables the determination of a cell line'sidentity. Placental and umbilicus-derived postpartum cells werecharacterized by flow cytometry for the expression of cell surfacemarkers CD10, CD13, CD31, CD34, CD44, CD45, CD73, CD90, CD117, CD141,PDGFr-alpha, HLA-A,B,C and HLA-DR, DP,DQ. Both placenta- andumbilicus-derived postpartum cells are positive for the expression ofCD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, B, C and negative forthe expression of CD31, CD34, CD44, CD45, CD73, CD90, CD117, CD141 andHLA-DR, DP, DQ. This expression pattern was consistent across variablessuch as cell donor, passage, culture vessel surface coating, anddigestion enzymes used in isolation. This expression pattern was alsoconsistent in cells isolated from the maternal aspect, neonatal aspectand villous region of the placenta.

Introduction

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum cell linesisolated from the placenta and umbilicus were characterized (by flowcytometry) providing a profile for the identification of these celllines.

Materials and Methods

Media

Cells were cultured in Growth Media.

Culture Vessels

Cells were cultured in plasma-treated T75, Ti50, and T225 tissue cultureflasks (Corning, Corning, N.Y.) until confluent. The growth surfaces ofthe flasks were coated with gelatin by incubating 2% (w/v) gelatin(Sigma, St. Louis, Mo.) for 20 minutes at room temperature.

Antibody Staining

Adherent cells in flasks were washed in phosphate buffered saline (PBS);(Gibco, Carlsbad, Mo.) and detached with Trypsin/EDTA (Gibco, Carlsbad,Mo.). Cells were harvested, centrifuged, and resuspended in 3% (v/v) FBSin PBS at a cell concentration of 1×10⁷ per milliliter. In accordance tothe manufacture's specifications, antibody to the cell surface marker ofinterest (see below) was added to one hundred microliters of cellsuspension and the mixture was incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were resuspended in 500 microliters PBSand analyzed by flow cytometry.

Flow Cytometry Analysis

Flow cytometry analysis was performed with a FACScalibur instrument(Becton Dickinson, San Jose, Calif.).

Antibodies to Cell Surface Markers

The following antibodies to cell surface markers were used. CatalogAntibody Manufacture Number CD10 BD Pharmingen 555375 (San Diego, CA)CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446 CD34 BD Pharmingen555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen 555489 CD73 BDPharmingen 550257 CD90 BD Pharmingen 555596 CD117 BD Pharmingen 340529CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen 556002 HLA-A, B, CBD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen 555558 IgG-FITC Sigma(St. Louis, MO) F-6522 IgG-PE Sigma P-4685

Placenta and Umbilicus Comparison

Placenta cells were compared to umbilicus at passage 8.

Passage to Passage Comparison

Placenta and umbilicus were analyzed at passages 8, 15, and 20.

Donor to Donor Comparison

To compare differences among donors, placenta cells from differentdonors were compared to each other, and umbilical from different donorswere compared to each other.

Surface Coating Comparison

Placenta cultured on gelatin-coated flasks was compared to placentacultured on uncoated flasks. Umbilicus cultured on gelatin-coated flaskswas compared to umbilicus cultured on uncoated flasks.

Digestion Enzyme Comparison

Four treatments used for isolation and preparation of cells werecompared. Cells isolated from placenta by treatment with 1) collagenase;2) collagenase/dispase; 3) collagenase/hyaluronidase; and 4)collagenase/hyaluronidase/dispase were compared.

Placental Layer Comparison

Cells isolated from the maternal aspect of placental tissue werecompared to cells isolated from the villous region of placental tissueand cells isolated from the neonatal fetal aspect of placenta.

Results

Placenta vs. Umbilicus Comparison

Placental- and umbilical-derived cells analyzed by flow cytometry showedpositive expression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha andHLA-A, B, C, indicated by the increased values of fluorescence relativeto the IgG control. These cells were negative for detectable expressionof CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated byfluorescence values comparable to the IgG control. Variations inflorescence values of positive curves was accounted for. The mean (i.e.CD13) and range (i.e. CD90) of the positive curves showed somevariation, but the curves appear normal, confirming a homogenouspopulation. Both curves individually exhibited values greater than theIgG control.

Passage to Passage Comparison—Placenta

Placenta-derived cells at passages 8, 15, and 20 analyzed by flowcytometry all were positive for expression of CD10, CD 13, CD44, CD73,CD90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased valueof fluorescence relative to the IgG control. The cells were negative forexpression of CD31, CD34, CD45, CD 117, CD 141, and HLA-DR, DP, DQhaving fluorescence values consistent with the IgG control. Variationsin florescence detection values of the positive curves was accountedfor. While range (i.e. CD10) of the positive curves varied, the curveswere normal, confirming a homogenous population, and each curvesindividually exhibited values greater than the IgG control.

Passage to Passage Comparison—Umbilicus

Umbilical cells at passage 8, 15, and 20 analyzed by flow cytometry allexpressed CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C,indicated by increased fluorescence relative to the IgG control. Thesecells were negative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP,DQ, indicated by fluorescence values consistent with the IgG control.Variations in florescence detection values of positive curves werewithin expected ranges. While the means (i.e. CD13) of the positivecurves varied all curves individually exhibited values greater than theIgG control.

Donor to Donor Comparison—Placenta

Placenta-derived cells isolated from separate donors analyzed by flowcytometry each expressed CD10, CD13, CD44, CD73, CD90, PDGFr-alpha andHLA-A, B, C, with increased values of fluorescence relative to the IgGcontrol. The cells were negative for expression of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence valueconsistent with the IgG control. Variations in florescence detectionvalues of positive curves are within expected ranges. While the range(i.e. CD44) of the positive curves varied, the curves appeared normal,confirming a homogenous population, and both curves individually exhibitvalues greater than the IgG control.

Donor to Donor Comparison—Umbilicus

Umbilical-derived cells isolated from separate donors analyzed by flowcytometry each showed positive expression of CD10, CD13, CD44, CD73,CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD 17, CD141, and HLA-DR, DP, DQ withfluorescence values consistent with the IgG control. Variations inflorescence detection values of positive curves were accounted for.While the mean (i.e. CD10) of the positive curves varied, both curvesindividually exhibited values greater than the IgG control.

The Effect of Surface Coating with Gelatin on Placenta-derived Cells

Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry all expressed of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ indicated by fluorescence values consistent with the IgG control.Variations in florescence detection values of positive curves werenoted. While the mean (i.e. PDGFr-alpha) of the positive curves variedboth curves individually exhibited values greater than the IgG control.

The Effect of Surface Coating with Gelatin on Umbilicus-derived Cells

Umbilical cells expanded on gelatin and uncoated flasks analyzed by flowcytometry all were positive for expression of CD10, CD13, CD44, CD73,CD90, PDGFr-alpha and HLA-A, B, C, with increased values of fluorescencerelative to the IgG control. These cells were negative for expression ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, with fluorescencevalues consistent with the IgG control.

Does the Enzyme Digestion Procedure Used for Preparation and Isolationof the Cells Effect the Cell Surface Marker Profile?

Placenta cells isolated using various digestion enzymes analyzed by flowcytometry all expressed CD10, CD13, CD44, CD73, CD90, PDGFr-alpha andHLA-A, B, C, as indicated by the increased values of fluorescencerelative to the IgG control. These cells were negative for expression ofCD31, CD34, CD45, CD 117, CD 141, and HLA-DR, DP, DQ as indicated byfluorescence values consistent with the IgG control. Variations in theCD13 mean florescence value were noted. While the CD 13 meanfluorescence values of the collagenase-treated cells was less than theother CD 13 curves, the collagenase-treated curve appeared normal,confirming a homogenous population, and it individually exhibited valuesgreater than the IgG control.

Placental Layer Comparison

Cells isolated from the maternal, villous, and neonatal layers of theplacenta, respectively, analyzed by flow cytometry showed positiveexpression of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C,as indicated by the increased value of fluorescence relative to the IgGcontrol. These cells were negative for expression of CD31, CD34, CD45,CD117, CD 141, and HLA-DR, DP, DQ as indicated by fluorescence valuesconsistent with the IgG control. Variations in florescence detectionvalues of positive curves were noted. While the mean and range (i.e.CD10, CD73) of the positive curves varied, both curves appeared normal,confirming a homogenous population, and all curves individually exhibitvalues greater than the IgG control.

CONCLUSIONS

Analysis of placenta- and umbilicus-derived postpartum cells by flowcytometry has established of an identity of these cell lines. Placenta-and umbilicus-derived postpartum cells are positive for CD10, CD 13,CD44, CD73, CD90, PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34,CD45, CD117, CD141 and HLA-DR, DP, DQ. This identity was consistentbetween variations in variables including the donor, passage, culturevessel surface coating, digestion enzymes, and placental layer. Somevariation in individual fluorescence value histogram curve means andranges were observed, but all positive curves under all conditionstested were normal and expressed fluorescence values greater than theIgG control, thus confirming that the cells comprise a homogenouspopulation which has positive expression of the markers.

EXAMPLE 6 Analysis of Postpartum Tissue-Derived Cells usingOligonucleotide Arrays

Oligonucleotide arrays were used to compare gene expression profiles ofumbilicus- and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Materials and Methods

Isolation and Culture of Cells

Postpartum tissue-derived cells. Human umbilical cords and placenta wereobtained from National Disease Research Interchange (NDRI, Philadelphia,Pa.) from normal full term deliveries with patient consent. The tissueswere received and cells were isolated as described in Example 1. Cellswere cultured in Growth Medium on gelatin-coated tissue culture plasticflasks. The cultures were incubated at 37° C. with 5% CO₂.

Fibroblasts. Human dermal fibroblasts were purchased from CambrexIncorporated (Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501(CCD39SK). Both lines were cultured in DMEM/F12 medium (Invitrogen,Carlsbad, Calif.) with 10% (v/v) fetal bovine serum (Hyclone) andpenicillin/streptomycin (Invitrogen). The cells were grown on standardtissue-treated plastic.

Human Mesenchymal Stem Cells (hMSC) hMSCs were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human Ileac Crest Bone Marrow Cells (ICBM). Human ileac crest bonemarrow was received from NDRI with patient consent. The marrow wasprocessed according to the method outlined by Ho, et al. (WO03/025149).The marrow was mixed with lysis buffer (155 millimolar NH₄Cl, 10millimolar KHCO₃, and 0.1 millimolar EDTA, pH 7.2) at a ratio of 1 partbone marrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 millimolar glutamine. The cellswere centrifuged again and the cell pellet was resuspended in freshmedium. The viable mononuclear cells were counted using trypan-blueexclusion (Sigma, St. Louis, Mo.). The mononuclear cells were seeded intissue-cultured plastic flasks at 5×10⁴ cells/cm². The cells wereincubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at5% O₂. Cells were cultured for 5 days without a media change. Media andnon-adherent cells were removed after 5 days of culture. The adherentcells were maintained in culture.

Isolation of mRNA and GENECHIP Analysis. Actively growing cultures ofcells were removed from the flasks with a cell scraper in cold phosphatebuffered saline (PBS). The cells were centrifuged for 5 minutes at300×g. The supernatant was removed and the cells were resuspended infresh PBS and centrifuged again. The supernatant was removed and thecell pellet was immediately frozen and stored at −80° C. Cellular mRNAwas extracted and transcribed into cDNA. cDNA was then transcribed intocRNA and biotin-labeled. The biotin-labeled cRNA was hybridized withAffymetrix GENECHIP HG-U133A oligonucleotide arrays (Affymetrix, SantaClara Calif.). The hybridizations and data collection were performedaccording to the manufacturer's specifications. The hybridization anddata collection was performed according to the manufacturer'sspecifications. Data analyses were performed using “SignificanceAnalysis of Microarrays” (SAM) version 1.21 computer software (Tusher,V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121). Licensesfor the analysis software are available through the Office of TechnologyLicensing, Stanford University, and more information is available on theWorld Wide Web at Professor Tibshirani's web site in the Dep't ofStatistics, Stanford University (www-stat.stanford.edu/˜tibs/SAM/).

Results

Fourteen different populations of cells were analyzed in this study. Thecells along with passage information, culture substrate, and culturemedia are listed in Table 6-1. TABLE 6-1 Cells analyzed by themicroarray study. The cells lines are listed by their identificationcode along with passage at the time of analysis, cell growth substrate,and growth media. Cell Population Passage Substrate Media Umbilical(022803) 2 Gelatin DMEM, 15% FBS, βME Umbilical (042103) 3 Gelatin DMEM,15% FBS, βME Umbilical (071003) 4 Gelatin DMEM, 15% FBS, βME Placenta(042203) 12 Gelatin DMEM, 15% FBS, βME Placenta (042903) 4 Gelatin DMEM,15% FBS, βME Placenta (071003) 3 Gelatin DMEM, 15% FBS, βME ICBM(070203) (5% O₂) 3 Plastic MEM 10% FBS ICBM (062703) (std O₂) 5 PlasticMEM 10% FBS ICBM (062703) (5% O₂) 5 Plastic MEM 10% FBS hMSC (Lot2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656) 3 Plastic MSCGM hMSC (Lot2F1657) 3 Plastic MSCGM hFibroblast (9F0844) 9 Plastic DMEM-F12, 10% FBShFibroblast (CCD39SK) 4 Plastic DMEM-F12, 10% FBS

The data were evaluated by Principle Component Analysis. Analysisrevealed 290 genes that were expressed in different relative amounts inthe cells tested. This analysis provided relative comparisons betweenthe populations.

Table 6-2 shows the Euclidean distances that were calculated for thecomparison of the cell pairs. The Euclidean distances were based on thecomparison of the cells based on the 290 genes that were differentiallyexpressed among the cell types. The Euclidean distance is inverselyproportional to similarity between the expression of the 290 genes.TABLE 6-2 The Euclidean Distances for the Cell Pairs. The Euclideandistance was calculated for the cell types using the 290 genes that wereexpressed differently between the cell types. Similarity between thecells is inversely proportional to the Euclidean distance. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 6-3,6-4, and 6-5 show the expression of genes increased inplacenta-derived cells (Table 6-3), increased in umbilical cord-derivedcells (Table 6-4), and reduced in umbilical cord and placenta-derivedcells (Table 6-5). TABLE 6-3 Genes which are specifically increased inexpression in the placenta-derived cells as compared to the other celllines assayed. Genes Increased in Placenta-Derived Cells NCBI AccessionProbe Set ID Gene Name Number 209732_at C-type (calcium dependent,AF070642 carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase AB015228 1 family, member A2 206367_at ReninNM_000537 210004_at oxidized low density AF035776 lipoprotein(lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE: AF0706424179671, mRNA, partial cds 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;AI246730 cDNA DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 6-4 Genes which are specifically increased in expression inumbilical cord-derived cells as compared to the other cell linesassayed. Genes Increased in Umbilicus-Derived Cells NCBI Accession ProbeSet ID Gene Name Number 202859_x_at Interleukin 8 NM_000584 211506_s_atInterleukin 8 AF043337 210222_s_at reticulon 1 BC000314 204470_atchemokine (C-X-C motif) NM_001511 ligand 1 (melanoma growth stimulatingactivity 206336_at chemokine (C-X-C motif) NM_002993 ligand 6(granulocyte chemotactic protein 2) 207850_at Chemokine (C-X-C motif)ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumornecrosis factor, NM_006290 alpha-induced protein 3

TABLE 6-5 Genes which were decreased in expression in the umbilical cordand placenta cells as compared to the other cell lines assayed. GenesDecreased in Umbilicus- and Placenta-Derived Cells Probe Set NCBIAccession ID Gene name Number 210135_s_at short stature homeobox 2AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_atchemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)U19495.1 203666_at chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1) NM_000609.1 212670_at elastin (supravalvularaortic stenosis, Williams-Beuren syndrome) AA479278 213381_at Homosapiens mRNA; cDNA DKFZp586M2022 (from clone N91149 DKFZp586M2022)206201_s_at mesenchyme homeobox 2 (growth arrest-specific homeobox)NM_005924.1 205817_at Sine oculis homeobox homolog 1 (Drosophila)NM_005982.1 209283_at crystallin, alpha B AF007162.1 212793_atdishevelled associated activator of morphogenesis 2 BF513244 213488_atDKFZP586B2420 protein AL050143.1 209763_at similar to neuralin 1AL049176 205200_at Tetranectin (plasminogen binding protein) NM_003278.1205743_at src homology three (SH3) and cysteine rich domain NM_003149.1200921_s_at B-cell translocation gene 1, anti-proliferative NM_001731.1206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_atrunt-related transcription factor 3 AA541630 219747_at hypotheticalprotein FLJ23191 NM_024574.1 204773_at Interleukin 11 receptor, alphaNM_004512.1 202465_at Procollagen C-endopeptidase enhancer NM_002593.2203706_s_at Frizzled homolog 7 (Drosophila) NM_003507.1 212736_athypothetical gene BC008967 BE299456 214587_at Collagen, type VIII, alpha1 BE877796 201645_at Tenascin C (hexabrachion) NM_002160.1 210239_atiroquois homeobox protein 5 U90304.1 203903_s_at Hephaestin NM_014799.1205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicleglycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis,clone MAMMA1001744 AU147799 206315_at cytokine receptor-like factor 1NM_004750.1 204401_at potassium intermediate/small conductancecalcium-activated channel, NM_002250.1 subfamily N, member 4 216331_atintegrin, alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1213125_at DKFZP586L151 protein AW007573 202133_at transcriptionalco-activator with PDZ-binding motif (TAZ) AA081084 206511_s_at Sineoculis homeobox homolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034protein AB028957.1 206115_at early growth response 3 NM_004430.1213707_s_at distal-less homeobox 5 NM_005221.3 218181_s_at hypotheticalprotein FLJ20373 NM_017792.1 209160_at aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid AB018580.1 dehydrogenase, type II)213905_x_at Biglycan AA845258 201261_x_at Biglycan BC002416.1 202132_attranscriptional co-activator with PDZ-binding motif (TAZ) AA081084214701_s_at fibronectin 1 AJ276395.1 213791_at Proenkephalin NM_006211.1205422_s_at Integrin, beta-like 1 (with EGF-like repeat domains)NM_004791.1 214927_at Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE AL359052.1 1968422 206070_s_at EphA3 AF213459.1 212805_atKIAA0367 protein AB002365.1 219789_at natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic AI628360 peptide receptor C)219054_at hypothetical protein FLJ14054 NM_024563.1 213429_at Homosapiens mRNA; cDNA DKFZp564B222 (from clone AW025579 DKFZp564B222)204929_s_at vesicle-associated membrane protein 5 (myobrevin)NM_006634.1 201843_s_at EGF-containing fibulin-like extracellular matrixprotein 1 NM_004105.2 221478_at BCL2/adenovirus E1B 19 kDa interactingprotein 3-like AL132665.1 201792_at AE binding protein 1 NM_001129.2204570_at cytochrome c oxidase subunit VIIa polypeptide 1 (muscle)NM_001864.1 201621_at neuroblastoma, suppression of tumorigenicity 1NM_005380.1 202718_at Insulin-like growth factor binding protein 2, 36kDa NM_000597.1

Tables 6-6,6-7, and 6-8 show the expression of genes increased in humanfibroblasts (Table 6-6), ICBM cells (Table 6-7), and MSCs (Table 6-8).TABLE 6-6 Genes which were increased in expression in fibroblasts ascompared to the other cell lines assayed. Genes increased in fibroblastsdual specificity phosphatase 2 KIAA0527 protein Homo sapiens cDNA:FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic, intermediatepolypeptide 1 ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta A(activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 6-7 Genes which were increased in expression in the ICBM-derivedcells as compared to the other cell lines assayed. Genes Increased InICBM Cells cardiac ankyrin repeat protein MHC class I region ORFintegrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptideN-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-inducedprotein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia,autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like1 jagged 1 (Alagille syndrome) proteoglycan 1, secretory granule

TABLE 6-8 Genes which were increased in expression in the MSC cells ascompared to the other cell lines assayed. Genes Increased In MSC Cellsinterleukin 26 maltase-glucoamylase (alpha-glucosidase) nuclear receptorsubfamily 4, group A, member 2 v-fos FBJ murine osteosarcoma viraloncogene homolog hypothetical protein DC42 nuclear receptor subfamily 4,group A, member 2 FBJ murine osteosarcoma viral oncogene homolog B WNT1inducible signaling pathway protein 1 MCF.2 cell line derivedtransforming sequence potassium channel, subfamily K, member 15cartilage paired-class homeoprotein 1 Homo sapiens cDNA FLJ12232 fis,clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone LIVER2000775jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc finger protein 51) zincfinger protein 36, C3H type, homolog (mouse)

Summary

The present study was performed to provide a molecular characterizationof the postpartum cells derived from umbilical cord and placenta. Thisanalysis included cells derived from three different umbilical cords andthree different placentas. The study also included two different linesof dermal fibroblasts, three lines of mesenchymal stem cells, and threelines of ileac crest bone marrow cells. The mRNA that was expressed bythese cells was analyzed on an GENECHIP oligonucleotide array thatcontained oligonucleotide probes for 22,000 genes.

The analysis revealed that transcripts for 290 genes were present indifferent amounts in these five different cell types. These genesinclude ten genes that are specifically increased in theplacenta-derived cells and seven genes specifically increased in theumbilical cord-derived cells. Fifty-four genes were found to havespecifically lower expression levels in placenta and umbilical cord.

The expression of selected genes has been confirmed by PCR (see Example7). These results demonstrate that the postpartum-derived cells have adistinct gene expression profile, for example, as compared to bonemarrow-derived cells and fibroblasts.

EXAMPLE 7 Cell Markers in Postpartum-Derived Cells

Summary

To examine cells derived from the human placenta and the human umbilicalcord, their gene expression profiles were compared to those of cellsderived from other sources using the Affymetrix GENECHIP. Through thistechnique we identified 6 genes that were highly expressed in postpartumcells: oxidized LDL receptor 1, interleukin-8, renin, reticulon,chemokine receptor ligand 3 (CXC ligand 3) and granulocyte chemotacticprotein 2 (GCP-2). Four of these genes (oxidized LDL receptor 1, renin,reticulon and IL-8) were differentially regulated at the mRNA level inpostpartum cells. IL-8 was found to also be differentially regulated atthe protein level.

We also investigated the expression of vimentin and alpha-smooth muscleactin, which has previously been associated with stroma-derived cells.Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theresult suggests that vimentin and alpha-smooth muscle actin expressionin cells is preserved with passaging in the Growth Medium.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of vimentin and alpha-smooth muscle actin proteins andwere positive for alpha-smooth muscle actin and vimentin with thepotential staining pattern preservation through passage 11.

The mRNA data at least partially verify the data obtained from themicroarray experiments.

Introduction

Similarities and differences in cells derived from the human placentaand the human umbilical cord were assessed by comparing their geneexpression profiles with those of cells derived from other sources(using an Affymetrix GENECHIP). Six “signature” genes were identified:oxidized LDL receptor 1, interleukin-8, renin, reticulon, chemokinereceptor ligand 3 (CXC ligand 3), and granulocyte chemotactic protein 2(GCP-2). These “signature” genes were expressed at relatively highlevels in postpartum-derived cells.

The present studies were conducted to verify the microarray data anddetermine correlations between gene and protein expression, as well asto establish a series of reliable assays for detection of uniqueidentifiers for placenta- and umbilical cord-derived cells.

Methods & Materials

Cells

Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis), umbilicalcord-derived cells (four isolates), and Normal Human Dermal Fibroblasts(NHDF; neonatal and adult) grown in Growth Medium in gelatin-coated T75flasks. Mesenchymal Stem Cells (MSCs) were grown in Mesenchymal StemCell Growth Medium Bullet kit (MSCGM; Cambrex, Walkerville, Md.).

For IL-8 experiment, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth Medium and then grown for further 8 hours in 10 milliliters ofserum starvation medium [DMEM —low glucose (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco, Carlsbad, Calif.) and 0.1% (w/v) BovineSerum Albumin (BSA; Sigma, St. Louis, Mo.)]. After this treatment RNAwas extracted and the supernatants were centrifuged at 150×g for 5minutes to remove cellular debris. Supernatants were then frozen at −80°C. for ELISA analysis.

Cell Culture for ELISA Assay.

Postpartum cells derived from placenta and umbilical cord, as well ashuman fibroblasts derived from human neonatal foreskin were cultured inGrowth Medium in gelatin-coated T75 flasks. Cells were frozen at passage11 in liquid nitrogen. Cells were thawed and transferred to 15 mlcentrifuge tubes. After centrifugation at 150×g for 5 minutes, thesupernatant was discarded. Cells were resuspended in 4 ml culture mediumand counted. Cells were grown in a 75 cm² flask containing 15 ml ofGrowth Medium at 375,000 cells/flask for 24 hours. The medium waschanged to a serum starvation medium for 8 hours. Serum starvationmedium was collected at the end of incubation, centrifuged at 14,000×gfor 5 minutes (and stored at −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added each flask. After cellsdetached from the flask, trypsin activity was neutralized with 8milliliters of Growth Medium. Cells were transferred to a 15 milliliterscentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth Medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA Assay

The amount of IL-8 secreted by the cells into serum starvation mediumwas analyzed using ELISA assays (R&D Systems, Minneapolis, Minn.). Allassays were tested according to the instructions provided by themanufacture.

Total RNA Isolation

RNA was extracted from confluent postpartum-derived cells andfibroblasts or for IL-8 expression from cells treated as describedabove. Cells were lysed with 350 microliters buffer RLT containingbeta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50microliters DEPC-treated water and stored at −80° C.

Reverse Transcription

RNA was also extracted from human placenta and umbilical cord. Tissue(30 mg) was suspended in 700 microliters of buffer RLT containingbeta-mercaptoethanol. Samples were mechanically homogenized and the RNAextraction proceeded according to manufacturer's specification. RNA wasextracted with 50 microliters of DEPC-treated water and stored at −80°C. RNA was reversed transcribed using random hexamers with the TaqManreverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95° C. for 10minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (signature genes—including oxidized LDL receptor, interleukin-8,renin and reticulon), were further investigated using real-time andconventional PCR.

Real-Time PCR

PCR was performed on cDNA samples using Assays-on-Demand™ geneexpression products: oxidized LDL receptor (Hs00234028); renin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH (Applied Biosystems, FosterCity, Calif.) were mixed with cDNA and TaqMan Universal PCR master mixaccording to the manufacturer's instructions (Applied Biosystems, FosterCity, Calif.) using a 7000 sequence detection system with ABI Prism 7000SDS software (Applied Biosystems, Foster City, Calif.). Thermal cycleconditions were initially 50° C. for 2 minutes and 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute.PCR data were analyzed according to manufacturer's specifications (UserBulletin #2 from Applied Biosystems for ABI Prism 7700 SequenceDetection System).

Conventional PCR

Conventional PCR was performed using an ABI PRISM 7700 (Perkin ElmerApplied Biosystems, Boston, Mass., USA) to confirm the results fromreal-time PCR. PCR was performed using 2 microliters of cDNA solution,1×AmpliTaq Gold universal mix PCR reaction buffer (Applied Biosystems,Foster City, Calif.) and initial denaturation at 94° C. for 5 minutes.Amplification was optimized for each primer set. For IL-8, CXC ligand 3,and reticulon (94° C. for 15 seconds, 55° C. for 15 seconds and 72° C.for 30 seconds for 30 cycles); for renin (94° C. for 15 seconds, 53° C.for 15 seconds and 72° C. for 30 seconds for 38 cycles); for oxidizedLDL receptor and GAPDH (94° C. for 15 seconds, 55° C. for 15 seconds and72° C. for 30 seconds for 33 cycles). Primers used for amplification arelisted in Table 7-1. Primer concentration in the final PCR reaction was1 μM except for GAPDH which was 0.5 μM. GAPDH primers were the same asreal-time PCR, except that the manufacturer's TaqMan probe was not addedto the final PCR reaction. Samples were run on 2% (w/v) agarose gel andstained with ethidium bromide (Sigma, St. Louis, Mo.). Images werecaptured using a 667 Universal Twinpack film (VWR International, SouthPlainfield, N.J.) using a focal-length Polaroid™ camera (VWRInternational, South Plainfield, N.J.). TABLE 7-1 Primers used Primername Primers Oxidized LDL receptor S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ (SEQID NO: 1) A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) Renin S:5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′(SEQ ID NO: 4) Reticulon S: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5)A: 5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO: 6) Interleukin-8 S:5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7) A:5′-CTTCAAAAACTTCTCCACAACC-3′ (SEQ ID NO: 8) Chemokine (CXC) ligand 3 S:5′-CCCACGCCACGCTCTCC-3′ (SEQ ID NO: 9) A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQID NO: 10)

Immunofluorescence

Postpartum cells were fixed with cold 4% (w/v) paraformaldehyde(Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at room temperature. Oneisolate each of umbilical- and placental-derived cells at passage 0 (P0)(directly after isolation) and passage 11 (P11) (two isolates ofPlacenta-derived, two isolates of Umbilical cord-derived cells) andfibroblasts (P11) were used. Immunocytochemistry was performed usingantibodies directed against the following epitopes: vimentin (1:500,Sigma, St. Louis, Mo.), desmin (1:150; Sigma—raised against rabbit; or1:300; Chemicon, Temecula, Calif.—raised against mouse,), alpha-smoothmuscle actin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma),von Willebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 ClassIII; 1:100; DAKOCytomation, Carpinteria, Calif.). In addition, thefollowing markers were tested on passage 11 postpartum cells: anti-humanGROalpha —PE (1:100; Becton Dickinson, Franklin Lakes, N.J.), anti-humanGCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.), anti-humanoxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa Cruz Biotech), andanti-human NOGA-A (1:100; Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG-Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG-Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG-FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 μM DAPI(Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution (no 1° control). Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Preparation of Cells for FACS Analysis

Adherent cells in flasks were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspended3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. Onehundred microliter aliquots were delivered to conical tubes. Cellsstained for intracellular antigens were permeablized with Perm/Washbuffer (BD Pharmingen, San Diego, Calif.). Antibody was added toaliquots as per manufactures specifications and the cells were incubatedfor in the dark for 30 minutes at 4° C. After incubation, cells werewashed with PBS and centrifuged to remove excess antibody. Cellsrequiring a secondary antibody were resuspended in 100 microliters of 3%FBS. Secondary antibody was added as per manufactures specification andthe cells were incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged to remove excesssecondary antibody. Washed cells were resuspended in 0.5 milliliters PBSand analyzed by flow cytometry. The following antibodies were used:oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.).

FACS Analysis

Flow cytometry analysis was performed with FACScalibur (Becton DickinsonSan Jose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentas, adult and neonatalfibroblasts and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and renin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the ΔΔCT method and expressed on alogarithmic scale. Levels of reticulon and oxidized LDL receptorexpression were higher in umbilical cord-derived cells as compared toother cells. No significant difference in the expression levels of CXCligand 3 and GCP-2 were found between postpartum cells and controls. Theresults of real-time PCR were confirmed by conventional PCR. Sequencingof PCR products further validated these observations. No significantdifference in the expression level of CXC ligand 3 was found betweenpostpartum cells and controls using conventional PCR CXC ligand 3primers listed in Table 7-1.

The expression of the cytokine, IL-8 in postpartum cells is elevated inboth Growth Medium-cultured and serum-starved postpartum-derived cells.All real-time PCR data were validated with conventional PCR and bysequencing PCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, the highest amounts were detected in media derivedfrom umbilical cells and some isolates of placenta cells (Table 7-2). NoIL-8 was detected in medium derived from human dermal fibroblasts. TABLE7-2 IL-8 protein expression measured by ELISA Cell type IL-8 hFibro NDPlacenta Isolate 1 ND UMBC Isolate 1 2058.42 ± 144.67 Placenta Isolate 2ND UMBC Isolate 2 2368.86 ± 22.73  Placenta Isolate 3 (normal O₂) 17.27± 8.63 Placenta Isolate 3 (lowO₂, W/O 264.92 ± 9.88  BME)Results of the ELISA assay for interleukin-8 (IL-8) performed onplacenta-and umbilical cord-derived cells as well as human skinfibroblasts. Values are presented here are pg/million cells, n = 2, sem.ND: Not Detected

Placenta-derived cells were also examined for the expression of oxidizedLDL receptor, GCP-2 and GROalpha by FACS analysis. Cells tested positivefor GCP-2. Oxidized LDL receptor and GRO were not detected by thismethod.

Placental cells were also tested for the expression of selected proteinsby immunocytochemical analysis. Immediately after isolation (passage 0),cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilical cord-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

Placenta-derived and umbilical cord-derived cells at passage 11 werealso investigated by immunocytochemistry for the expression of GROalpha,GCP-2, oxidized LDL receptor 1 and reticulon. Complete results of thatexperiment are still pending.

DISCUSSION AND CONCLUSION

Thus far concordance between gene expression levels measured bymicroarray and PCR (both real-time and conventional) has beenestablished for four genes: oxidized LDL receptor 1, renin, reticulon,and IL-8. The expression of these genes was differentially regulated atthe mRNA level in postpartum cells, with IL-8 also differentiallyregulated at the protein level. The presence of oxidized LDL receptorwas not detected at the protein level by FACS analysis in cells derivedfrom the placenta. Differential expression of GCP-2 and CXC ligand 3 wasnot confirmed at the mRNA level, however GCP-2 was detected at theprotein level by FACS analysis in the placenta-derived cells.Differences between these data and that obtained from the microarrayexperiment may be due to differences in the sensitivity of themethodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin expressionmay be preserved in cells with passaging, at least in the Growth Mediumused here.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of alpha-smooth muscle actin and vimentin, and werepositive for both. The staining pattern was preserved through passage11.

In conclusion, the complete mRNA data at least partially verify the dataobtained from the microarray experiments. As additional proteinexperiments are complete, the relationships between mRNA and proteinexpression will be more comprehensively understood.

EXAMPLE 8 Immunohistochemical Characterization of PPDC Phenotype

The phenotypes of cells found within human postpartum tissues, namelyumbilical cord and placenta, were analyzed by immunohistochemistry.

Materials & Methods

Tissue Preparation. Human umbilical cord and placenta tissue washarvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at4° C. Immunohistochemistry was performed using antibodies directedagainst the following epitopes (see Table 8-1): vimentin (1:500; Sigma,St. Louis, Mo.), desmin (1:150, raised against rabbit; Sigma; or 1:300,raised against mouse; Chemicon, Temecula, Calif.), alpha-smooth muscleactin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), vonWillebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III;1:100; DAKOCytomation, Carpinteria, Calif.). In addition, the followingmarkers were tested: anti-human GROalpha —PE (1:100; Becton Dickinson,Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech,Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1;1:100; Santa Cruz Biotech), and anti-human NOGO-A (1:100; Santa CruzBiotech). Fixed specimens were trimmed with a scalpel and placed withinOCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on adry ice bath containing ethanol. Frozen blocks were then sectioned (10μm thick) using a standard cryostat (Leica Microsystems) and mountedonto glass slides for staining. TABLE 8-1 Summary of Primary AntibodiesUsed Antibody Concentration Vendor Vimentin 1:500 Sigma, St. Louis, MODesmin (rb) 1:150 Sigma Desmin (m) 1:300 Chemicon, Temecula, CA alphasmooth 1:400 Sigma muscle actin (SMA) Cytokeratin 18 1:400 Sigma (CK18)von Willebrand 1:200 Sigma factor (vWF) CD34 III 1:100 DakoCytomation,Carpinteria, CA GROalpha - PE 1:100 BD, Franklin Lakes, NJ GCP-2 1:100Santa Cruz Biotech Ox-LDL R1 1:100 Santa Cruz Biotech NOGO-A 1:100 SantaCruz Biotech

Immunohistochemistry. Immunohistochemistry was performed similar toprevious studies (e.g., Messina, et al. (2003) Exper. Neurol. 184:816-829). Tissue sections were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 1 hour to access intracellular antigens. Ininstances where the epitope of interest would be located on the cellsurface (CD34, ox-LDL R1), Triton was omitted in all steps of theprocedure in order to prevent epitope loss. Furthermore, in instanceswhere the primary antibody was raised against goat (GCP-2, ox-LDL R1,NOGO-A), 3% (v/v) donkey serum was used in place of goat serumthroughout the procedure. Primary antibodies, diluted in blockingsolution, were then applied to the sections for a period of 4 hours atroom temperature. Primary antibody solutions were removed, and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing block along with goat anti-mouseIgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goatanti-rabbit IgG-Alexa 488 (1:250; Molecular Probes) or donkey anti-goatIgG-FITC (1:150; Santa Cruz Biotech). Cultures were washed, and 10 μMDAPI (Molecular Probes) was applied for 10 minutes to visualize cellnuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color video camera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results

Umbilical Cord Characterization. Vimentin, desmin, SMA, CK18, vWF, andCD34 markers were expressed in a subset of the cells found withinumbilical cord. In particular, vWF and CD34 expression were restrictedto blood vessels contained within the cord. CD34+cells were on theinnermost layer (lumen side). Vimentin expression was found throughoutthe matrix and blood vessels of the cord. SMA was limited to the matrixand outer walls of the artery & vein, but not contained with the vesselsthemselves. CK18 and desmin were observed within the vessels only,desmin being restricted to the middle and outer layers.

Placenta Characterization. Vimentin, desmin, SMA, CK18, vWF, and CD34were all observed within the placenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression. None of thesemarkers were observed within umbilical cord or placental tissue.

Summary. Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18,von Willebrand Factor, and CD34 are expressed in cells within humanumbilical cord and placenta.

EXAMPLE 9 Secretion of Trophic Factors by Postpartum Cells Derived FromPlacenta and Umbilical Cord

The secretion of selected trophic factors from placenta- and umbilicalcord-derived PPDCs was measured. Factors were selected that haveangiogenic activity (i.e., hepatocyte growth factor (HGF) (Rosen et al.(1997) Ciba Found. Symp. 212:215-26), monocyte chemotactic protein 1(MCP-1) (Salcedo et al. (2000) Blood 96;34-40), interleukin-8 (IL-8) (L1et al. (2003) J. Immunol. 170:3369-76), keratinocyte growth factor(KGF), basic fibroblast growth factor (bFGF), vascular endothelialgrowth factor (VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8),matrix metalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2), plateletderived growth factor (PDGF-bb), thrombopoietin (TPO), heparin-bindingepidermal growth factor (HB-EGF), stromal-derived factor 1 alpha (SDF-1alpha)), neurotrophic/neuroprotective activity (brain-derivedneurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol. 258;319-33),interleukin-6 (IL-6), granulocyte chemotactic protein-2 (GCP-2),transforming growth factor beta2 (TGFbeta2)), or chemokine activity(macrophage inflammatory protein 1 alpha (MIP1a), macrophageinflammatory protein 1beta (MIP1b), monocyte chemoattractant-1 (MCP-1),Rantes (regulated on activation, normal T cell expressed and secreted),I309, thymus and activation-regulated chemokine (TARC), Eotaxin,macrophage-derived chemokine (MDC), IL-8).

Methods & Materials

Cell culture. PPDCs derived from placenta and umbilical cord as well ashuman fibroblasts derived from human neonatal foreskin were cultured inGrowth Medium on gelatin-coated T75 flasks. Cells were cryopreserved atpassage 11 and stored in liquid nitrogen. After thawing of the cells,Growth Medium was added to the cells followed by transfer to a 15milliliters centrifuge tube and centrifugation of the cells at 150×g for5 minutes. The supernatant was discarded. The cell pellet wasresuspended in 4 milliliters Growth Medium, and cells were counted.Cells were seeded at 375,000 cells/75 cm² flask containing 15milliliters of Growth Medium and cultured for 24 hours. The medium waschanged to a serum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v)bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) for 8hours. Conditioned serum-free media was collected at the end ofincubation by centrifugation at 14,000×g for 5 minutes and stored at−20° C. To estimate the number of cells in each flask, cells were washedwith phosphate-buffered saline (PBS) and detached using 2 milliliterstrypsin/EDTA (Gibco). Trypsin activity was inhibited by addition of 8milliliters Growth Medium. Cells were centrifuged at 150×g for 5minutes. Supernatant was removed, and cells were resuspended in 1milliliter Growth Medium. Cell number was estimated using ahemocytometer.

ELISA assay. Cells were grown at 37° C. in 5% carbon dioxide andatmospheric oxygen. Placenta-derived PPDCs (101503) also were grown in5% oxygen or beta-mercaptoethanol (BME). The amount of MCP-1, IL-6,VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta2 produced by each cellsample was measured by an ELISA assay (R&D Systems, Minneapolis, Minn.).All assays were performed according to the manufacturer's instructions.Values presented are pg/ml/million cells (n=2, sem).

SearchLight Multiplexed ELISA assay. Chemokines (MIP1a, MIP1b, MCP-1,Rantes, I309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors(HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF were measuredusing SearchLight Proteome Arrays (Pierce Biotechnology Inc.). TheProteome Arrays are multiplexed sandwich ELISAs for the quantitativemeasurement of two to 16 proteins per well. The arrays are produced byspotting a 2×2, 3×3, or 4×4 pattern of four to 16 different captureantibodies into each well of a 96-well plate. Following a sandwich ELISAprocedure, the entire plate is imaged to capture chemiluminescent signalgenerated at each spot within each well of the plate. The amount ofsignal generated in each spot is proportional to the amount of targetprotein in the standard or sample.

Results

ELISA assay. MCP-1 and IL-6 were secreted by placenta- and umbilicalcord-derived PPDCs and dermal fibroblasts (Table 9-1). SDF-1alpha wassecreted by placenta-derived PPDCs cultured in 5% O₂ and by fibroblasts.GCP-2 and IL-8 were secreted by umbilical-derived PPDCs and byplacenta-derived PPDCs cultured in the presence of BME or 5% O₂. GCP-2also was secreted by human fibroblasts. TGF-beta2 was not detectable byELISA assay. TABLE 9-1 ELISA assay results TGF- MCP-1 IL-6 VEGF SDF-lαGCP-2 IL-8 β2 Fibroblast 17 ± 1  61 ± 3  29 ± 2 19 ± 1 21 ± 1 ND NDPlacenta (042303) 60 ± 3  41 ± 2  ND ND ND ND ND Umbilical (022803) 1150± 74   4234 ± 289  ND ND 160 ± 11  2058 ± 145 ND Placenta (071003) 125 ±16  10 ± 1  ND ND ND ND ND Umbilical (071003) 2794 ± 84  1356 ± 43  NDND  2184 ± 98  2369 ± 23 ND Placenta (101503) BME 21 ± 10 67 ± 3  ND ND44 ± 9  17 ± 9  ND Placenta (101503) 5% O₂, W/O 77 ± 16 339 ± 21  ND1149 ± 137 54 ± 2  265 ± 10 ND BMEKey: ND: Not Detected.

SearchLight Multiplexed ELISA assay. TIMP1, TPO, KGF, HGF, FGF, HBEGF,BDNF, MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were secreted fromumbilical cord-derived PPDCs (Tables 9-2 and 9-3). TIMP1, TPO, KGF, HGF,HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were secretedfrom placenta-derived PPDCs (Tables 9-2 and 9-3). No Ang2, VEGF, orPDGF-bb were detected. TABLE 9-2 SearchLight Multiplexed ELISA assayresults TIMP1 ANG2 PDGFbb TPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 NDND 230.5 5.0 ND ND 27.9 1.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND3.81.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7 ND 9.3 165.7 P314176.8 ND ND 568.7 5.2 10.2 ND ND 1.9 33.6 U3 21850.0 ND ND 1134.5 9.0195.6 30.8 ND 5.4 388.6Key: hFB (human fibroblasts), P1 (placenta-derived PPDC (042303)), U1(umbilical cord-derived PPDC (022803)), P3 (placenta-derived PPDC(071003)), U3 (umbilical cord-derived PPDC (071003)). ND: Not Detected.

TABLE 9-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4 4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.511.6 21.4 ND 4.8 10515.9Key: hFB (human fibroblasts), P1 (placenta-derived PPDC (042303)), U1(umbilical cord-derived PPDC (022803)), P3 (placenta-derived PPDC(071003)), U3 (umbilical cord-derived PPDC (071003)). ND: Not Detected.

Summary. Umbilical cord- and placenta-derived cells secreted a number oftrophic factors. Some of these trophic factors, such as HGF, bFGF, MCP-1and IL-8, play important roles in angiogenesis. Other trophic factors,such as BDNF and IL-6, have important roles in neural regeneration.

EXAMPLE 10 In vitro Immunology

Postpartum cell lines were evaluated in vitro for their immunologicalcharacteristics in an effort to predict the immunological response, ifany, these cells would elicit upon in vivo transplantation. Postpartumcell lines were assayed by flow cytometry for the expression of HLA-DR,HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4⁺ T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the expressionof HLA-G (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed.(2003) Saunders, Philadelphia, p. 171), CD178 (Coumans, et. al., (1999)Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas &Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders,Philadelphia, p. 171; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The expression of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to which postpartumplacenta- and umbilical cord-derived cell lines elicit an immuneresponse in vivo, the cell lines were tested in a one-way mixedlymphocyte reaction (MLR).

Materials and Methods

Cell Culture. Cells were cultured in Growth Media until confluent in T75flasks (Corning, Corning, N.Y.) coated with 2% gelatin (Sigma, St.Louis, Mo.).

Antibody Staining. Cells were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Mo.). Cells were harvested, centrifuged, and re-suspended in3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter.Antibody (Table 10-1) was added to one hundred microliters of cellsuspension as per manufacturer's specifications and incubated in thedark for 30 minutes at 4° C. After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were re-suspendedin five hundred microliters of PBS and analyzed by flow cytometry usinga FACSCalibur instrument (Becton Dickinson, San Jose, Calif.). TABLE10-1 Antibodies Antibody Manufacture Catalog Number HLA-DRDPDQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA)557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen(San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100CD178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse Sigma (St.Louis, MO) P-4685 IgG1kappa

Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10 umbilicalcord-derived PPDCs labeled as cell line A and passage 11placenta-derived PPDCs labeled as cell line B were sent on dry ice toCTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction usingCTBR SOP no. CAC-031. Peripheral blood mononuclear cells (PBMCs) werecollected from multiple male and female volunteer donors. Stimulator(donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines weretreated with mitomycin C. Autologous and mitomycin C-treated stimulatorcells were added to responder (recipient) PBMCs and cultured for 4 days.After incubation, [³H]thymidine was added to each sample and culturedfor 18 hours. Following harvest of the cells, radiolabeled DNA wasextracted, and [³H]-thymidine incorporation was measured using ascintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the postpartum cell was calculated as the meanproliferation of the receiver plus mitomycin C-treated postpartum cellline divided by the baseline proliferation of the receiver.

Results

Mixed Lymphocyte Reaction-Placenta. Seven human volunteer blood donorswere screened to identify a single allogeneic donor that would exhibit arobust proliferation response in a mixed lymphocyte reaction with theother six blood donors. This donor was selected as the allogeneicpositive control donor. The remaining six blood donors were selected asrecipients. The allogeneic positive control donor and placenta celllines were treated with mitomycin C and cultured in a mixed lymphocytereaction with the six individual allogeneic receivers. Reactions wereperformed in triplicate using two cell culture plates with threereceivers per plate (Table 10-2). The average stimulation index rangedfrom 1.3 (plate 2) to 3 (plate 1) and the allogeneic donor positivecontrols ranged from 46.25 (plate 2) to 279 (plate 1) (Table 10-3).TABLE 10-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPMfor Proliferation Assay Analytical Culture Replicates number System 1 23 Mean SD CV Plate ID: Plate 1 IM03-7769 Proliferation baseline ofreceiver 79 119 138 112.0 30.12 26.9 Control of autostimulation(Mitomycin C treated autologous cells) 241 272 175 229.3 49.54 21.6 MLRallogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 2092122414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B)664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Controlof autostimulation (Mitomycin C treated autologous cells) 1091 602 524739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C treated)45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin Ctreated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI(cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128124.0 35.17 28.4 Control of autostimulation (Mitomycin C treatedautologous cells) 293 138 508 313.0 185.81 59.4 MLR allogenic donorIM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8MLR with cell line (Mitomycin C treated cell type B) 601 643 a 622.029.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferationbaseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation(Mitomycin C treated autologous cells) 133 120 213 155.3 50.36 32.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 2216818822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell typeB) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferationbaseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic donor)Control of autostimulation (Mitomycin treated autologous cells) 361 617304 427.3 166.71 39.0 Cell line type B Proliferation baseline ofreceiver 126 124 143 131.0 10.44 8.0 Control of autostimulation(Mitomycin treated autologous cells) 822 1075 487 794.7 294.95 37.1Plate ID: Plate 2 IM03-7773 Proliferation baseline of receiver 908 181330 473.0 384.02 81.2 Control of autostimulation (Mitomycin C treatedautologous cells) 269 405 572 415.3 151.76 36.5 MLR allogenic donorIM03-7768 (Mitomycin C treated) 29151 28691 28315 28719.0 418.70 1.5 MLRwith cell line (Mitomycin C treated cell type B) 567 732 905 734.7169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774 Proliferationbaseline of receiver 893 1376 185 818.0 599.03 73.2 Control ofautostimulation (Mitomycin C treated autologous cells) 261 381 568 403.3154.71 38.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 5310142839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C treatedcell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI (cell line)1 IM03-7775 Proliferation baseline of receiver 1272 300 544 705.3 505.6971.7 Control of autostimulation (Mitomycin C treated autologous cells)232 199 484 305.0 155.89 51.1 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 23554 10523 28965 21014.0 9479.74 45.1 MLR with cell line(Mitomycin C treated cell type B) 768 924 563 751.7 181.05 24.1 SI(donor) 30 SI (cell line) 1 IM03-7776 Proliferation baseline of receiver1530 137 1046 904.3 707.22 78.2 Control of autostimulation (Mitomycin Ctreated autologous cells) 420 218 394 344.0 109.89 31.9 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.229.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI(donor) 35 SI (cell line) a

TABLE 10-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers. Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

Mixed Lymphocyte Reaction— Umbilicus. Six human volunteer blood donorswere screened to identify a single allogeneic donor that will exhibit arobust proliferation response in a mixed lymphocyte reaction with theother five blood donors. This donor was selected as the allogeneicpositive control donor. The remaining five blood donors were selected asrecipients. The allogeneic positive control donor and placenta celllines were mitomycin C-treated and cultured in a mixed lymphocytereaction with the five individual allogeneic receivers. Reactions wereperformed in triplicate using two cell culture plates with threereceivers per plate (Table 10-4). The average stimulation index rangedfrom 6.5 (plate 1) to 9 (plate 2) and the allogeneic donor positivecontrols ranged from 42.75 (plate 1) to 70 (plate 2) (Table 10-5). TABLE10-4 Mixed Lymphocyte Reaction Data- Cell Line A (Umbilicus) DPM forProliferation Assay Analytical Culture Replicates number System 1 2 3Mean SD CV Plate ID: Plate 1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic donor) Control of autostimulation (Mitomycin treatedautologous cells) 567 604 374 515.0 123.50 24.0 Cell line type AProliferation baseline of receiver 5101 3735 2973 3936.3 1078.19 27.4Control of autostimulation (Mitomycin treated autologous cells) 19244570 2153 2882.3 1466.04 50.9

TABLE 10-5 Average stimulation index of umbilical cells and anallogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Recipient UmbilicusPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

Antigen Presenting Cell Markers—Placenta. Histograms of placenta cellsanalyzed by flow cytometry show negative expression of HLA-DR, DP, DQ,CD80, CD86, and B7-H2, as noted by fluorescence value consistent withthe IgG control, indicating that placental cell lines lack the cellsurface molecules required to directly stimulate CD4⁺ T cells.

Immuno-modulating Markers—Placenta. Histograms of placenta cellsanalyzed by flow cytometry show positive expression of PD-L2, as notedby the increased value of fluorescence relative to the IgG control, andnegative expression of CD178 and HLA-G, as noted by fluorescence valueconsistent with the IgG control.

Antigen Presenting Cell Markers—Umbilicus. Histograms of umbilical cellsanalyzed by flow cytometry show negative expression of HLA-DR, DP, DQ,CD80, CD86, and B7-H2, as noted by fluorescence value consistent withthe IgG control, indicating that umbilical cell lines lack the cellsurface molecules required to directly stimulate CD4⁺ T cells.

Immuno-modulating Markers—Umbilicus. Histograms of umbilical cellsanalyzed by flow cytometry show positive expression of PD-L2, as notedby the increased value of fluorescence relative to the IgG control, andnegative expression of CD178 and HLA-G, as noted by fluorescence valueconsistent with the IgG control.

Summary. In the mixed lymphocyte reactions conducted with placental celllines, the average stimulation index ranged from 1.3 to 3, and that ofthe allogeneic positive controls ranged from 46.25 to 279. In the mixedlymphocyte reactions conducted with umbilical cell lines the averagestimulation index ranged from 6.5 to 9, and that of the allogeneicpositive controls ranged from 42.75 to 70. Placental and umbilical celllines were negative for the expression of the stimulating proteinsHLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flowcytometry. Placental and umbilical cell lines were negative for theexpression of immuno-modulating proteins HLA-G and CD 178 and positivefor the expression of PD-L2, as measured by flow cytometry. Allogeneicdonor PBMCs contain antigen-presenting cells expressing HLA-DR, DP, DQ,CD80, CD86, and B7-H2, thereby allowing for the stimulation of naïveCD4⁺ T cells. The absence of antigen-presenting cell surface moleculeson placenta- and umbilical cord-derived cells required for the directstimulation of naïve CD4⁺ T cells and the presence of PD-L2, animmuno-modulating protein, may account for the low stimulation indexexhibited by these cells in a MLR as compared to allogeneic controls.

EXAMPLE 11 Plasma Clotting Assay

Cell therapy may be injected systemically for certain applications wherecells are able to target the site of action. It is important thatinjected cells not cause thrombosis, which may be fatal. Tissue factor,a membrane-bound procoagulant glycoprotein, is the initiator of theextrinsic clotting cascade, which is the predominant coagulation pathwayin vivo. Tissue factor also plays an important role in embryonic vesselformation, for example, in the formation of the primitive vascular wall(Brodsky et al. (2002) Exp. Nephrol. 10:299-306). To determine thepotential for PPDCs to initiate clotting, umbilical cord andplacenta-derived PPDCs were evaluated for tissue factor expression andtheir ability to initiate plasma clotting.

Methods & Materials

Human Tissue factor. SIMPLASTIN, a human tissue factor (Organon TekailcaCorporation, Durham, N.C.), was reconstituted with 20 millilitersdistilled water. The stock solution was serially diluted (1:2) in eighttubes. Normal human plasma (George King Bio-Medical, Overland Park,Kans.) was thawed at 37° C. in a water bath and then stored in icebefore use. To each well of a 96-well plate was added 100 microlitersphosphate buffered saline (PBS), 10 microliters diluted SIMPLASTIN(except a blank well), 30 microliters 0.1M calcium chloride, and 100microliters of normal human plasma. The plate was immediately placed ina temperature-controlled microplate reader and absorbance measured at405 nanometers at 40 second intervals for 30 minutes.

J-82 and postpartum cells. J-82 cells (ATCC, MD) were grown in Iscove'smodified Dulbecco's medium (IMDM; Gibco, Carlsbad, Calif.) containing10% (v/v) fetal bovine serum (FBS; Hyclone, Logan Utah), 1 millimolarsodium pyruvate (Sigma Chemical, St. Louis, Mo.), 2 millimolarL-Glutamine (Mediatech Herndon, Va.), 1×non-essential amino acids(Mediatech Herndon, VA). At 70% confluence, cells were transferred towells of 96-well plate at 100,000, 50,000 and 25,000 cells/well.Postpartum cells derived from placenta and umbilical cord were culturedin Growth Medium in gelatin-coated T75 flasks (Corning, Corning, N.Y.).Placenta-derived cells at passage 5 and umbilical cord-derived cells atpassages 5 and 18 were transferred to wells at 50,000 cells/well.Culture medium was removed from each well after centrifugation at 150×gfor 5 minutes. Cells were suspended in PBS without calcium andmagnesium. Cells incubated with anti-tissue factor antibody cells wereincubated with 20 micrograms/milliliter CNTO 859 (Centocor, Malvern,Pa.) for 30 minutes. Calcium chloride (30 microliters) was added to eachwell. The plate was immediately placed in a temperature-controlledmicroplate reader and absorbance measured at 405 nanometers at 40 secondintervals for 30 minutes.

Antibody Staining. Cells were washed in PBS and detached from the flaskwith Trypsin/EDTA (Gibco Carlsbad, Calif.). Cells were harvested,centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. Antibody was added to 100microliters cell suspension as per the manufacturer's specifications,and the cells were incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged at 150×g for 5minutes to remove unbound antibody. Cells were re-suspended in 100microliters of 3% FBS and secondary antibody added as per themanufacturer's instructions. Cells were incubated in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove unbound secondary antibody. Washed cells werere-suspended in 500 microliters of PBS and analyzed by flow cytometry.

Flow Cytometry Analysis. Flow cytometry analysis was performed with aFACSCalibur instrument (Becton Dickinson, San Jose, Calif.).

Results

Flow cytometry analysis revealed that both placenta- and umbilicalcord-derived postpartum cells express tissue factor. A plasma clottingassay demonstrated that tissue factor was active. Both placenta- andumbilical cord-derived cells increased the clotting rate as indicated bythe time to half maximal absorbance (T ½to max; Table 11-1). Clottingwas observed with both early (P5) and late (P18) cells. The T ½ to maxis inversely proportional to the number of J82 cells. Preincubation ofumbilical cells with CNTO 859, an antibody to tissue factor, inhibitedthe clotting reaction, thereby showing that tissue factor wasresponsible for the clotting. TABLE 11-1 The effect of human tissuefactor (SIMPLASTIN), placenta- derived cells (Pla), and umbilicalcord-derived cells (Umb) on plasma clotting was evaluated. The time tohalf maximal absorbance (T ½ to max) at the plateau in seconds was usedas a measurement unit. SIMPLASTIN Dilution T ½ to max (seconds) 1:2 611:4 107 1:8 147 1:16 174 1:32 266 1:64 317 1:128 378 0 (negativecontrol) 1188 J-82 cells 100,000 122  50,000 172  25,000 275 Pla P5 50,000 757 Umb P5  50,000 833 Umb P18  50,000 443

Summary. Placenta- and umbilical cord-derived PPDCs express tissuefactor. The addition of an antibody to tissue factor can inhibit tissuefactor. Tissue factor is normally found on cells in a conformation thatis inactive but is activated by mechanical or chemical (e.g., LPS)stress (Sakariassen et al. (2001) Thromb. Res. 104:149-74; Engstad etal. (2002) Int. Immunopharmacol. 2:1585-97). Thus, minimization ofstress during the preparation process of PPDCs may prevent activation oftissue factor. In addition to the thrombogenic activity, tissue factorhas been associated with angiogenic activity. Thus, tissue factoractivity may be beneficial when umbilical cord- or placenta-derivedPPDCs are transplanted in tissue but should be inhibited when PPDCs areinjected intravenously.

FIG. 11-1. Plasma clotting with J82 cells and umbilical cells without(A) and with (B) CNTO 859, anti-human tissue factor, 20 μg/milliliters.Antibody treatment showed that coagulation was due to tissue factor onumbilical cells.

EXAMPLE 12 Differentiation of Postpartum Cells to the CardiomyocytePhenotype

There is a tremendous need for therapy that will slow the progression ofand/or cure heart disease, such as ischemic heart disease and congestiveheart failure. Cells that can differentiate into cardiomyocytes that canfully integrate into the patient's cardiac muscle without arrhythmias ishighly desirable. Rodent mesenchymal stem cells treated with5-azacytidine have been shown to express markers of cardiomyocytes(Fukuda et al. (2002) C. R. Biol. 325:1027-38). The same has not beenshown for adult human stem cells. Additional factors have been used toimprove stem cell differentiation including low oxygen (Storch (1990)Biochim. Biophys. Acta 1055:126-9), retinoic acid (Wobus et al. (1997)J. Mol. Cell Cardiol. 29:1525-39), DMSO (Xu et al. (2002) Circ. Res.91:501-8), and chelerythrine chloride (International PCT Publication No.WO03/025149), which effects the translocation of PKC from the cytosol toplasma membrane and is an inhibitor of PKC activity. In the presentstudy, PPDCs (P10) were treated with 5-azacytidine either alone or incombination with DMSO or chelerythrine chloride and markers ofcardiomyocytes measured by real-time PCR.

Methods & Materials

Cells. Cryopreserved umbilical cord-derived cells (P10) andplacenta-derived cells (P24) were grown in Growth Medium ingelatin-coated flasks. Cells were seeded at 5×10⁴ cells/well in 96-wellplates in Growth Medium for 24 hours. The medium was changed to 0, 3, 10and 30 μM 5-azacytidine (Sigma, St. Louis, Mo.) alone or with 5 μMchelerythrine chloride (Sigma), 1% (v/v) dimethylsulfoxide (DMSO)(Sigma), or 1 μM retinoic acid (Sigma) in MEM-alpha (Sigma), insulin,transferrin, and selenium (ITS; Sigma), 10% (v/v) fetal bovine serum,penicillin and streptomycin, and cells incubated at 37° C., 5% (v/v) O₂for 48 or 72 hours. Media was then changed to MEM-alpha, insulin,transferrin, and selenium, 10% (v/v) fetal bovine serum, penicillin andstreptomycin, and cells incubated at 37° C., 5% (v/v) O₂ for 14 days.

RNA extraction and Reverse Transcription. Cells were lysed with 150microliters buffer RLT containing beta-mercaptoethanol (Sigma St. Louis,Mo.) according to the manufacturer's instructions (RNeasy 96 kit,Qiagen, Valencia, Calif.) and stored at −80° C. Cell lysates were thawedand RNA extracted according to the manufacturer's instructions (RNeasy96 kit, Qiagen, Valencia, Calif.) with a 2.7 U/sample DNase treatment(Sigma St. Louis, Mo.). RNA was eluted with 50 microliters DEPC-treatedwater and stored at −80° C. RNA was reverse transcribed using randomhexamers with the TaqMan reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes and 95° C. for 10 minutes. Samples were stored at −20° C.

PCR. PCR was performed on cDNA samples using Assays-on-Demand™ geneexpression products cardiac myosin (Hs00165276 ml), skeletal myosin(Hs00428600), GATA 4 (Hs00171403 ml), GAPDH (Applied Biosystems, FosterCity, Calif.), and TaqMan Universal PCR master mix according to themanufacturer's instructions (Applied Biosystems, Foster City, Calif.)using a 7000 sequence detection system with ABI prism 7000 SDS software(Applied Biosystems, Foster City, Calif.). Thermal cycle conditions wereinitially 50° C. for 2 minutes and 95° C. for 10 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute. cDNA from heartand skeletal muscle (Ambion Austin Tex.) were used as a control.

Results

Analysis of RNA from cardiac muscle showed expression of cardiac myosinand GATA 4. Skeletal muscle RNA analysis showed expression skeletalmyosin and cardiac myosin but not of GATA 4. Placenta-derived cells(P24) treated for 72 h with factors and grown for a further14 daysexpressed GATA 4, but no skeletal myosin or cardiac myosin. Umbilicalcord-derived cells (P12) treated for 48h with factors and cultured for afurther 14 days expressed low levels of GATA 4, but no skeletal myosinor cardiac myosin. Additional samples from placenta and umbilical cordcells that were analyzed showed expression of GATA 4 under theconditions tested.

Summary. Untreated placenta- and umbilical cord-derived cellsconstitutively express GATA 4, a nuclear transcription factor incardiomyocytes, sertoli cells, and hepatocytes.

EXAMPLE 13 Assessment of Postpartum Cell-Based Cardiovascular Therapy ina Rodent Coronary Ligation Model

Animal models of heart failure have increased our understanding of thepathophysiology of the disease and have assisted in the development ofnew treatments for congestive heart failure (CHF). Coronary arteryocclusion, or the blocking of the vessels that supply the heart tissue,in the rat closely mimics the pathophysiology of acute myocardialinfarction in humans and has been used successfully to studypharmacological interventions for CHF. Transplantation of human cellsinto cardiac lesions is a potential viable therapeutic treatment forCHF.

The objective of this study was to determine the efficacy ofintracardiac human cell treatment when administered 15 minutespost-coronary artery occlusion in a rodent model of myocardialischemia/infarction.

Methods & Materials

The Charles River Worcester, Mass. test facility is accredited by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare, International (AAALAC) and registered with the United StatesDepartment of Agriculture to conduct research in laboratory animals. Allthe conditions of testing conform to the Animal Welfare Act (9 CFR) andits amendments. The protocol was reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) at the Test Facilityfor compliance with regulations prior to study initiation.

The animals having characteristics identified in Table 13-1 wereindividually housed in micro-isolator cages on autoclaved bedding. Thecages conform to standards set forth in The Guide for the Care and Useof Laboratory Animals. TABLE 13-1 Animal characteristics Species: Rattusnorvegicus Strain: Rnu Source: Charles River Laboratories Age at Dosing:6-8 weeks Weight at Dosing: ˜200-250 g Number of Males (includingspares): 40 + 10

Purina Certified Diet (irradiated) was provided to the animals adlibitum. This diet was routinely analyzed by the manufacturer fornutritional components and environmental contaminants. Results of themanufacturer's analyses are on file at the Test Facility.

Autoclaved Filtered tap water was provided ad libitum. Samples of thefiltered water were analyzed for total dissolved solids, hardness,specified microbiological content, and selected environmentalcontaminants. Results of these analyses are on file at the TestFacility.

Environmental controls were set to maintain temperatures of 18 to 26° C.(64 to 79° F.) with a relative humidity of 30% to 70%. A 12:12 hourlight:dark cycle was maintained. Ten or greater air changes per hourwere maintained in the animal rooms. Upon receipt and prior to use onthe study, the animals were held for a minimum of four days forconditioning according to the Test Facility Vendor Management Program asdescribed in the Test Facility Standard Operating Procedure, Receipt,Conditioning, and Quarantine of Laboratory Animals.

Each animal was identified by a unique number and this number wasindicated by an ear punch. Animals were randomly assigned to groups by aweight-ordered distribution such that individual body weights did notexceed ±20% of mean weight.

The animals were anesthetized with sodium pentobarbital (40milligrams/kilogram) and buprenorphine(0.05 milligrams/kilogram) as asingle cocktail given intramuscularly (IM). Following the establishmentof anesthesia, animals were intubated using an 18-16 gauge, 2-inchlength angiocath, or appropriate sized angiocath, and maintained on roomair respiration (supplemented with oxygen) and a positive pressureventilator throughout the surgical procedure. Additional anesthesia wasgiven incrementally as needed. Preoperative antibiotic therapy was alsoadministered, Benzathine/Procaine penicillin G, 40,000 Units/kilogram,IM. Additional antibiotic therapy was administered every 48 hours.

Electrode pads were placed around the appropriate paws of the animals toreceive a useable ECG signal. Animals were positioned on a heating padto help maintain body temperature throughout the procedure. A rectaltemperature probe was inserted into the animal to monitor bodytemperature. Ophthalmic ointment was administered to each eye. Thesurgical sites (thoracic area) were prepared for aseptic surgery byremoving any excess fur, and gently wiping the area with sponges thathave been soaked in 70% isopropyl alcohol, which was allowed to dry.Medi Sepps™ or similar solution was then applied to the area and alsoallowed to dry. The area was appropriately draped for strict asepticsurgery.

A surgical incision was made on the skin over the fourth intercostalspace. Blunt dissection through the muscle layers was used to access thethoracic cavity. A retractor was carefully inserted into the fourthintercostal space and opened to allow access to the interior cavity. Thepericardium was carefully opened via gentle teasing with cotton swabsdampened in sterile saline solution. A damp cotton swab was used togently push the apex of the heart into the opening where a length of 6-0silk suture was attached into the myocardium for manipulation of theheart. After a pause to allow the heart to recover, the suture placed inthe apex was used to ease the heart out of the chest cavity and to placesufficient tension on the heart to allow access to the upper heart andthe left anterior descending coronary artery (LAD). Another length of6-0 silk suture was placed into the myocardium so as to surround theLAD. The pressure on the apical suture was released and the heartallowed to return to the interior of the chest cavity.

Once the heart rate and ECG returned to baseline values, the ligaturesaround the LAD were tied off to occlude the LAD. This was a permanentocclusion with the suture tied off and the ends trimmed. Once theligature was tied, the surgeon looked for the following indications ofsuccessful occlusion: change in color of the area of the heart directlybelow the ligature to a white/grayish white as a result of thetermination of blood flow to the area and a significant change in theECG corresponding to occlusion of the LAD. Arrhythmias may havedeveloped within the first 10 minutes of the occlusion. The rat wasmonitored closely during this time period in the event thatresuscitation was necessary. In the event of severe arrhythmia andfailure of the rat to convert to normal sinus rhythm without assistance,aid was rendered via cardiac massage. Approximately 15 minutes followingthe initiation of the LAD occlusion, the area of left ventricle madeischemic was treated with either vehicle or test article by directinjection into the ischemic myocardium. Treatment consisted of three toten intramyocardial injections (100 microliters/injection) into theischemic zone of myocardium.

Human cells were grown in Growth Medium in gelatin-coated T300 flasks.Cells were washed with phosphate buffered saline (PBS, Gibco, CarlsbadCalif.) and trypsinized using Trypsin/EDTA (Gibco, Carlsbad Calif.). Thetrypsinization was stopped by adding Growth Medium. The cells werecentrifuged at 150×g, supernatant removed, and the cell pellet wasresuspended in approximately 1 milliliter Growth Medium per millioncells. An aliquot of cells was removed and added to trypan blue (Sigma,St. Louis, Mo.). The viable cell number was estimated using ahemocytometer. The cell suspension was centrifuged and resuspended in 1milliliters Growth containing 10% (v/v) DMSO (Hybrimax, Sigma, St.Louis, Mo.) per 5 million cells and transferred into Cryovials(Nalgene). The cells were cooled at approximately 1° C./min overnight ina −80° C. freezer using a “Mr Frosty” freezing container (Nalgene,Rochester, N.Y.). Vials of cells were transferred into liquid nitrogen.Vials were shipped from CBAT, Somerville, N.J. to Charles River,Worcester, Mass. on dry ice and stored at −80° C. Approximately 1-2hours before injection of cells into the animal, a vial of cells wasthawed rapidly in a 37° C. water bath. Under aseptic conditions in aBSL2 biosafety cabinet, cells were added to 40 milliliters PBS withmagnesium and calcium (Sigma St. Louis, Mo.) and centrifuged at 150×gfor 5 minutes before resuspending the cell pellet in 10 milliliters PBS.The cell number and viability was estimated as described above. Thecells were centrifuged at 150×g for 5 minutes and resuspended in PBS ata final concentration of 10⁶ viable cells/100 microliters. The cellsuspension was loaded into 1 milliliter syringes with a 30G needle andkept on ice. Viability was assessed again up to 5 hours on ice.

Following the administration of treatment (Table 13-2) and stabilizationof the heart, the surgeon began closing the surgical incision. Theretractor was removed. The lungs were over-inflated for 3-4 breaths andvisually inspected as much as possible to ensure that they were fullyre-inflated. This created a negative pressure necessary to preventpneumothorax post-recovery. To evacuate fluid and excess air from thethoracic cavity after closing the cavity, an intravenous catheter (i.e.,20 gauge, 2 mm in length) was placed through the skin and muscle layersso that the tip remains in the thoracic cavity. Care was taken so thatthe tip did not pierce the lung or heart. The separated ribs andassociated muscle was sutured together with appropriate suture. Theupper layers of muscle was sutured using a simple continuous pattern.The skin was closed with 4-0 silk using a horizontal mattress pattern. A10 milliliter syringe was attached to the intravenous catheter that hadbeen previously placed in the thoracic cavity and the plunger slowlypulled back to withdraw fluids and air from the cavity. At the sametime, the catheter was slowly withdrawn from the entry site, therebyallowing the surrounding muscle mass and skin to seal the puncture. Thesurgical drape was removed and fluids (i.e., lactated Ringers solution,25 milliliters/kilogram subcutaneously [SC] or intraperitoneally [IP])were given. TABLE 13-2 Treatment regimens Dosage Dose Conc. Time of Gr.No. of Level (cells/ Route/Dose Treatment Necropsy No. Males TestArticle (cells/animal) milliliters) Regimen Administration Day 1 8Vehicle 0 0 Direct 15 minutes Day 28 2 8 Placenta #4 (P10) 1 million 10million injection(s) into after coronary (±1 Day) (A) the ischemicartery ligation 3 8 Umbilical (P10) region of the left (B) ventricle ofthe 4 8 Placenta #3 (P10) heart, consisting (C) of 3 to 10 5 8 Humanintramyocardial fibroblasts injections of 1F1853 (P10) (D) 100 μL total.Gr. = Group; No. = Number; Conc. = Concentration

Immediately after each rat had undergone treatment with test article andthe incision sutured, the animal underwent an echocardiography (ECG)examination. Anesthesia was maintained throughout the completion of theecho examination. Upon the completion of the echo examination,ventilation was discontinued, and the rat was returned to the recoveryarea to recover in a heated, oxygenated recovery cage.

A second echo examination of each surviving animal was completed at theend of the study (approximately 28 days post-treatment), prior totermination. During the second examination, the animals wereanesthetized as described previously.

For each echo examination, the left thoracic area was shaved, andwarmed, ultrasonic gel was applied to the skin to enhance contact withthe transducer. Electrode pads were placed around the appropriateextremities to receive an ECG signal. Echocardiographic images includedshort axis and long axis views to allow for the determination ofventricular cavity dimensions, contractility, blood flow throughvasculature, and wall thickness. These images were saved on optical diskfor further analysis. After examination, the gel medium was removed fromthe skin with gauze or paper towel. The rat was removed from theventilator and placed in a warmed recovery cage until mobile.

At the conclusion of the surgical procedures, respiratory ventilationwas turned off. The animals were observed for pedal reflex. The rectalprobe and ECG electrodes subsequently were removed, and the animal wasextubated and placed in a warmed oxygenated recovery cage. Aftercomplete recovery from anesthesia, the animals were given buprenorphine(0.05 milligrams/kilogram, SC). Observations were made regularly untilthe animals showed full mobility and an interest in food and water. Theanimals then were placed in a clean housing cage and returned to theanimal housing room. Animals were monitored for surgical incisionintegrity twice daily post-surgery.

Analgesics (i.e., Buprenorphine, 0.05 milligrams/kilogram SC.) weregiven twice daily for 4 days post-operatively and thereafter as needed.Visual indications of post-operative pain include lack of normal bodypostures and movement (e.g., animal remains in hunched position),antipathy, lack of eating/drinking, lack of grooming, etc.

Body weight was recorded for each animal prior to initial treatment,weekly thereafter, and on the day of necropsy. Animals found dead wereweighed and necropsied.

In order for the heart to be harvested, each rat was anesthetized as wasdone for surgery. The jugular vein was cannulated. The heart wasarrested in diastole with KCl infused via the jugular cannula. The heartwas then removed from the thoracic cavity. A limited necropsy was thenperformed on the heart after which the heart was placed in 10% neutralbuffered formalin. The remainder of each carcass was then discarded withno further evaluation.

Hearts of all animals that were found dead or euthanized moribund wereplaced in 4% paraformaldehyde until evaluated. The remainder of eachcarcass was then discarded with no further evaluation.

Histology and Image Analysis. Fixed tissues sectioned with a stainlesssteel coronal heart matrix (Harvard Apparatus Holliston, Mass.) yieldedfour two-millimeter thick serial tissue sections. Sections wereprocessed and serially embedded in paraffin using routine methods.Five-micron sections were obtained by microtome and stained Masson'sTri-chrome for Connective Tissue (Poly Scientific Bay Shore, N.Y.) usingmanufacturer's methods. Electronic photomicrographs were captured andanalyzed using image analysis methods developed by Phase 3 ImagingSystem (Glen Mills, Pa.). Photomicrographs of the tri-chrome stainedsections were color-metrically analyzed electronically to determine theoverall area of the ventricle and free wall and the area of thedifferential staining.

Results

There was no loss in the initial viability of cells over 5 hours in thevehicle when kept on ice. Cells were injected into the infarct with oneto three needle entry points and multiple changes in direction of needleorientation.

Echocardiography measurements taken from the infarct-treated rats wereused to evaluate the results. The left ventricle fractional shorteningand the ejection fraction wee similarly utilized. These values werecalculated as described by Sahn et al. (1978) Circulation 58:1072-1083.The fractional shortening of the vehicle-treated animals wassignificantly decreased from 47.7%±8.3% at Day 0 to 23.5%±30.2% at Day28 (p<0.05). The animals that were treated with postpartum-derived cellsshowed small, non-significant differences between the fractionalshortening between Day 0 and 28. There was no significant differencebetween the fractional shortening between the groups at Day 0. Eachgroup had eight animals at the start but some did not survive theexperiment. The fibroblast-treated animals experienced greater mortality(80%) than the groups treated with PPDCs.

Hearts collected at the study termination were subjected to histologicalanalysis. The hearts were arrested in diastole and fixed. The resultswere calculated from an algorithm to estimate the percentage of totalheart area that comprises the infarct. The infarct size in thevehicle-treated animals was 22.9%±6.7% of heart area, while the infarctsize in hearts treated with placenta-derived cells (isolate 1) was13.9%±3.7%, with umbilical cord cells was 12.5%±2.5%, withplacenta-derived cells (isolate 2) was 12.9%±3.4%, and with fibroblastswas 19.3%±8.0%. The difference of infarct size of cell-treated animalsrelative to vehicle-treated animals was not statistically significant byeither Student's t test or ANOVA.

Summary. The results of the present study suggest that thepostpartum-derived cells may have some benefit in reducing the damage ofa surgically induced myocardial infarction in rats. The vehicle-treatedanimals showed a significant reduction in cardiac function at day 28 ascompared to day 0, as measured by fractional shortening, while theplacenta- and umbilical cord-derived cell-treated animals showed minimalchange over the 28-day study. The fibroblast-treated animals showedminimal change but only two animals survived the study. Evaluation ofinfarct size suggested that there may be some modest, but notstatistically significant, reduction in the infarct size in thepostpartum-derived cell-treated animals as compared to the vehiclecontrols at Day 28. Taken together, these data establish efficacy of thepostpartum-derived cells in reducing damage from myocardial infarction.

EXAMPLE 14 PPDCs Can Be Injected as a Matrix-Cell Complex

Cells derived from the postpartum umbilical cord are useful forregenerative therapies. The tissue produced by postpartum-derived cellstransplanted into SCID mice with a biodegradable material was evaluated.The materials evaluated were Vicryl non-woven, 35/65 PCL/PGA foam, andRAD 16 self-assembling peptide hydrogel.

Methods & Materials

Cell Culture. Umbilical cord-derived cells were grown in Growth Mediumin gelatin-coated flasks.

Sample Preparation. One million viable cells were seeded in 15microliters Growth Medium onto 5 mm diameter, 2.25 mm thick Vicrylnon-woven scaffolds (64.33 mg/cc; Lot#3547-47-1) or 5 mm diameter 35/65PCL/PGA foam (Lot# 3415-53). Cells were allowed to attach for two hoursbefore adding more Growth Medium to cover the scaffolds. Cells weregrown on scaffolds overnight. Scaffolds without cells were alsoincubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 millimolar HEPES in Dulbecco's modifiedmedium (DMEM; Gibco) immediately before use. The final concentration ofcells in RAD16 hydrogel was 1×10⁶ cells/100 microliters.

TESTMATERIAL (N=4/Rx)

-   -   1. Vicryl non-woven+1×10⁶ umbilical cord-derived cells    -   2. 35/65 PCL/PGA foam+1×10⁶ umbilical cord-derived cells    -   3. RAD 16 self-assembling peptide+1×10⁶ umbilical cord-derived        cells    -   4. 35/65 PCL/PGA foam    -   5. Vicryl non-woven

Animal Preparation. The animals utilized in this study were handled andmaintained in accordance with the current requirements of the AnimalWelfare Act. Compliance with the above Public Laws were accomplished byadhering to the Animal Welfare regulations (9 CFR) and conforming to thecurrent standards promulgated in the Guide for the Care and Use ofLaboratory Animals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 weeks of age. All handling of the SCID mice tookplace under a hood. The mice were individually weighed and anesthetizedwith an intraperitoneal injection of a mixture of 60 milligrams/kilogramKETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and10 milligrams/kilogram ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.)and saline. After induction of anesthesia, the entire back of the animalfrom the dorsal cervical area to the dorsal lumbosacral area was clippedfree of hair using electric animal clippers. The area was then scrubbedwith chlorhexidine diacetate, rinsed with alcohol, dried, and paintedwith an aqueous iodophor solution of 1% available iodine. Ophthalmicointment was applied to the eyes to prevent drying of the tissue duringthe anesthetic period.

Subcutaneous Implantation Technique. Four skin incisions, eachapproximately 1.0 cm in length, were made on the dorsum of the mice. Twocranial sites were located transversely over the dorsal lateral thoracicregion, about 5-mm caudal to the palpated inferior edge of the scapula,with one to the left and one to the right of the vertebral column.Another two were placed transversely over the gluteal muscle area at thecaudal sacro-lumbar level, about 5-mm caudal to the palpated iliaccrest, with one on either side of the midline. Implants were randomlyplaced in these sites. The skin was separated from the underlyingconnective tissue to make a small pocket and the implant placed (orinjected for RAD16) about 1-cm caudal to the incision. The appropriatetest material was implanted into the subcutaneous space. The skinincision was closed with metal clips.

Animal Housing. Mice were individually housed in microisolator cagesthroughout the course of the study within a temperature range of 64°F.-79° F. and relative humidity of 30% to 70%, and maintained on anapproximate 12 hour light/12 hour dark cycle. The temperature andrelative humidity were maintained within the stated ranges to thegreatest extent possible. Diet consisted of Irradiated Pico Mouse Chow5058 (Purina Co.) and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology. Excised skin with implant was fixed with 10% neutral bufferedformalin (Richard-Allan Kalamazoo, Mich.). Samples with overlying andadjacent tissue were centrally bisected, paraffin-processed, andembedded on cut surface using routine methods. Five-micron tissuesections were obtained by microtome and stained with hematoxylin andeosin (Poly Scientific Bay Shore, N.Y.) using routine methods.

Results

There was minimal in growth of tissue into foams implantedsubcutaneously in SCID mice after 30 days. In contrast there wasextensive tissue fill in foams implanted with umbilical-derived cells.

There was some tissue in growth in Vicryl non-woven scaffolds. Non-wovenscaffolds seeded with umbilical cord-derived cells showed increasedmatrix deposition and mature blood vessels.

Summary. The purpose of this study was to determine the type of tissueformed by cells derived from human umbilical cord in scaffolds in immunedeficient mice. Synthetic absorbable non-woven/foam discs (5.0 mmdiameter×1.0 mm thick) or self-assembling peptide hydrogel were seededwith cells derived from human umbilical cord and implantedsubcutaneously bilaterally in the dorsal spine region of SCID mice. Thepresent study demonstrates that postpartum-derived cells candramatically increase good quality tissue formation in biodegradablescaffolds.

EXAMPLE 15 Endothelial Network Formation: PPDCs Promote Angiogenesis InVitro

Angiogenesis, or the formation of new vasculature, is necessary for thegrowth of new tissue. Induction of angiogenesis is an importanttherapeutic goal in many pathological conditions. The present study wasaimed at identifying potential angiogenic activity of the postpartumcells in in vitro assays. The study followed a well-established methodof seeding endothelial cells onto a culture plate coated with a basementmembrane extract (Nicosia and Ottinetti (1990) In Vitro Cell Dev. Biol.26(2):119-28). Treating endothelial cells on extracellular matrix withangiogenic factors will stimulate the cells to form a network that issimilar to capillaries. This is a common in vitro assay for testingstimulators and inhibitors of blood vessel formation (Ito et al. (1996)Int. J. Cancer 67(1):148-52). The present studies made use of aco-culture system with the postpartum cells seeded onto culture wellinserts. The permeable inserts allow for the passive exchange of mediacomponents between the endothelial and the postpartum culture media.

Material & Methods

Cell Culture.

Postpartum tissue-derived cells. Human umbilical cords and placenta werereceived and cells were isolated as previously described (Example 1).Cells were cultured in Growth Medium on gelatin-coated tissue cultureplastic flasks. The cultures were incubated at 37° C. with 5% CO₂. Cellsused for experiments were from about passage 4 to 12.

Actively growing postpartum cells were trypsinized, counted, and seededonto Costar® Transwell® 6.5 mm diameter tissue culture inserts (Corning,Corning, N.Y.) at 15,000 cells per insert. Cells were cultured on theinserts for 48-72 hours in Growth Medium at 37° C. with 5% CO₂.

Human mesenchymal stem cells (hMSC). hMSCs were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). The cultures wereincubated at 37° C. with 5% CO₂.

Actively growing MSCs were trypsinized, counted and seeded onto Costar®Transwell® 6.5 mm diameter tissue culture inserts (Corning, Corning,N.Y.) at 15,000 cells per insert. Cells were cultured on the inserts for48-72 hours in Growth Medium at 37° C. with 5% CO₂.

Human umbilical vein endothelial cells. (HUVEC). HUVEC were obtainedfrom Cambrex (Walkersville, Md.). Cells were grown in separate culturesin either EBM or EGM endothelial cell media (Cambrex). Cells were grownon standard tissue cultured plastic at 37° C. with 5% CO₂. Cells used inthe assay were from about passage 4 to 10.

Human coronary artery endothelial cells (HCAEC). HCAEC were purchasedfrom Cambrex Incorporated (Walkersville, Md.). The cells were maintainedin separate cultures in either the EBM or EGM media formulations. Cellswere grown on standard tissue culture plastic at 37° C. with 5% CO₂.Cells used for experiments were from about passage 4 to 8.

Matrigel™ assays. Culture plates were coated with Matrigel™ according tomanufacturer's specifications. Briefly, Matrigel™ (BD Discovery Labware,Bedford, Mass.) was thawed at 4° C. and approximately 250 microliterswas aliquoted and distributed evenly onto each well of a chilled 24-wellculture plate (Corning). The plate was then incubated at 37° C. for 30minutes to allow the material to solidify. Actively growing endothelialcell cultures were trypsinized and counted. Cells were washed twice inGrowth Media with 2% FBS by centrifugation, resuspension, and aspirationof the supernatant. Cells were seeded onto the coated wells 20,000 cellsper well in approximately 0.5 milliliters Growth Medium with 2% (v/v)FBS. Cells were then incubated for approximately 30 minutes to allowcells to settle.

Endothelial cell cultures were then treated with either 10 nanomolarhuman bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanomolar human VEGF(Peprotech, Rocky Hill, N.J.) to serve as a positive control forendothelial cell response. Transwell inserts seeded with postpartumcells were added to appropriate wells with Growth media with 2% FBS inthe insert chamber. Cultures were incubated at 37° C. with 5% CO₂ forapproximately 24 hours. The well plate was removed from the incubator,and images of the endothelial cell cultures were collected with anOlympus inverted microscope (Olympus, Melville, N.Y.).

Results

In a co-culture system with placenta-derived cells or with umbilicalcord-derived cells, HUVEC form cell networks. HUVEC cells form limitedcell networks in co-culture experiments with hMSC and with 10 nanomolarbFGF. HUVEC cells without any treatment showed very little or no networkformation. These results suggest that the postpartum cells releaseangiogenic factors that stimulate the HUVEC.

In a co-culture system with placenta-derived cells or with umbilicalcord-derived cells, CAECs form cell networks.

Table 15-1 shows levels of known angiogenic factors released by thepostpartum cells in Growth Medium. Postpartum cells were seeded ontoinserts as described above. The cells were cultured at 37° C. inatmospheric oxygen for 48 hours on the inserts and then switched to a 2%FBS media and returned at 37° C. for 24 hours. Media were removed,immediately frozen and stored at −80° C., and analyzed by theSearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford,Ill.). Results shown are the averages of duplicate measurements. Theresults show that the postpartum cells do not release detectable levelsof platelet-derived growth factor-bb (PDGF-bb) or heparin-bindingepidermal growth factor (HBEGF). The cells do release measurablequantities of tissue inhibitor of metallinoprotease-1 (TIMP-1),angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor(KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF),and vascular endothelial growth factor (VEGF). TABLE 15-1 Potentialangiogenic factors released from postpartum cells. Postpartum cells werecultured in 24 hours in media with 2% FBS in atmospheric oxygen. Mediawere removed and assayed by the SearchLight multiplex ELISA assay(Pierce). Results are the means of a duplicate analysis. Values areconcentrations in the media reported in picograms per milliliter ofculture media. TIMP1 ANG2 PDGFBB TPO KGF HGF FGF VEGF HBEGF (pg/mL)(pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) Placenta91655.3 175.5 <2.0 275.5 3.0 58.3 7.5 644.6 <1.2 (P4) Placenta 1592832.428.1 <2.0 1273.1 193.3 5960.3 34.8 12361.1 1.7 (P11) Umbilical 81831.7<9.8 <2.0 365.9 14.1 200.2 5.8 <4.0 <1.2 cord (P4) Media alone <9.8 25.1<2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2

Table 15-2 shows levels of known angiogenic factors released by thepostpartum cells. Postpartum cells were seeded onto inserts as describedabove. The cells were cultured in Growth Medium at 5% oxygen for 48hours on the inserts and then switched to a 2% FBS medium and returnedto 5% O₂ incubation for 24 hours. Media were removed, immediatelyfrozen, and stored at −80° C., and analyzed by the SearchLight multiplexELISA assay (Pierce Chemical Company, Rockford, Ill.). Results shown arethe averages of duplicate measurements. The results show that thepostpartum cells do not release detectable levels of platelet-derivedgrowth factor-bb (PDGF-BB), or heparin-binding epidermal growth factor(HBEGF). The cells do release measurable quantities of tissue inhibitorof metallinoprotease-1 (TIMP-1), angiopoietin 2 (ANG2), thrombopoietin(TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF),fibroblast growth factor (FGF) and vascular endothelial growth factor(VEGF). TABLE 15-2 Potential angiogenic factors released from postpartumcells. Postpartum cells were cultured in 24 hours in media with 2% FBSin 5% oxygen. Media were removed and assayed by the SearchLightmultiplex ELISA assay (Pierce). Results are the means of a duplicateanalysis. Values are concentrations in the media reported in picogramsper milliliter of culture media. TIMP1 ANG2 PDGFBB TPO KGF HGF FGF VEGFHBEGF (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL)(pg/mL) Placenta 72972.5 253.6 <2.0 743.1 2.5 30.2 15.1 1495.1 <1.2 (P4)Placenta 458023.1 55.1 <2.0 2562.2 114.2 2138.0 295.1 7521.3 1.8 (P11)Umbilical 50244.7 <9.8 <2.0 403.3 10.7 156.8 5.7 <4.0 <1.2 cord (P4)Media alone <9.8 25.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2

Summary. Postpartum cells can stimulate both human umbilical vein andcoronary artery endothelial cells to form networks in an in vitroMatrigel™ assay. This effect is similar to that with known angiogenicfactors in this assay system, suggesting that the postpartum cells areuseful for stimulating angiogenesis in vivo.

Biological Deposit of Postpartum-Derived Cells and Cultures

Consistent with the detailed description and the written examplesprovided herein, examples of umbilicus-derived cells of the inventionwere deposited with the American Type Culture Collection (ATCC,Manassas, Va.) on Jun. 10, 2004, and assigned ATCC Accession Numbers asfollows: (1) strain designation UMB 022803 (P7) was assigned AccessionNo. PTA-6067; and (2) strain designation UMB 022803 (P17) was assignedAccession No. PTA-6068.

As with the umbilicus-derived cells, examples of placenta-derived cellsof the invention were also deposited with the American Type CultureCollection (ATCC, Manassas, Va.) and assigned ATCC Accession Numbers asfollows: (1) strain designation PLA 071003 (P8) was deposited Jun. 15,2004 and assigned Accession No. PTA-6074; (2) strain designation PLA071003 (P11) was deposited June 15, 2004 and assigned Accession No.PTA-6075; and (3) strain designation PLA 071003 (P16) was deposited Jun.16, 2004 and assigned Accession No. PTA-6079.

1. An isolated postpartum-derived cell comprising an L-valine-requiringcell derived from human postpartum tissue substantially free of blood,said cell capable of self-renewal and expansion in culture and havingthe potential to differentiate into a cell of cardiomyocyte phenotypes;the cell capable of growth in an atmosphere containing oxygen from about5% to at least about 20%; wherein said cell comprises at least one ofthe following characteristics: potential for at least about 40 doublingsin culture; attachment and expansion on a coated or uncoated tissueculture vessel, wherein a coated tissue culture vessel comprises acoating of gelatin, laminin, collagen, polyornithine, vitronectin, orfibronectin; production of at least one of tissue factor, vimentin, andalpha-smooth muscle actin; production of at least one of CD10, CD13,CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; lack of productionof at least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178,B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry;expression of at least one of interleukin 8; reticulon 1; chemokine(C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha);chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2);chemokine (C-X-C motif) ligand 3; and tumor necrosis factor,alpha-induced protein 3; expression of at least one of C-type (calciumdependent, carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced); Wilms tumor 1; aldehyde dehydrogenase 1 family,member A2; and renin; oxidized low density lipoprotein (lectin-like)receptor 1; Homo sapiens, clone IMAGE:4179671, mRNA, partial cds;protein kinase C, zeta; hypothetical protein DKFZp564F013; downregulatedin ovarian cancer 1; Homo sapiens mRNA; and cDNA DKFZp547K1113 (fromclone DKFZp547K1113); expression, which relative to a human cell that isa fibroblast, a mesenchymal stem cell, or an ileac crest bone marrowcell, is reduced for at least one of: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeobox 2 (growth arrest-specifichomeobox); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; dishevelled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;B-cell translocation gene 1, anti-proliferative; cholesterol25-hydroxylase; runt-related transcription factor 3; hypotheticalprotein FLJ23191; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, alpha 7; DKFZP586L151 protein; transcriptionalco-activator with PDZ-binding motif (TAZ); sine oculis homeobox homolog2 (Drosophila); KIAA1034 protein; early growth response 3; distal-lesshomeobox 5; hypothetical protein FLJ20373; aldo-keto reductase family 1,member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-likerepeat domains); Homo sapiens mRNA full length insert cDNA cloneEUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide receptorC/guanylate cyclase C (atrionatriuretic peptide receptor C);hypothetical protein FLJ14054; Homo sapiens mRNA; cDNA DKFZp564B222(from clone DKFZp564B222); vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; secretion of at least one of MCP-1,IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, andTIMP1; and lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb,MIP1b, I309, MDC, and VEGF, as detected by ELISA.
 2. Thepostpartum-derived cell of claim 1 isolated in the presence of one ormore enzyme activities comprising metalloprotease activity, mucolyticactivity and neutral protease activity.
 3. The postpartum-derived cellof claim 2 wherein the enzyme activities are collagenase and dispase. 4.The postpartum-derived cell of claim 3 further comprising hyaluronidase.5. The postpartum-derived cell of claim 4 comprising a normal karyotype.6. The postpartum-derived cell of claim 5 that maintains its karyotypeas it is passaged.
 7. The postpartum-derived cell of claim 6 wherein thecell expresses each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A,B,C.
 8. The postpartum-derived cell of claim 7 wherein the celldoes not express any of CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ,as detected by flow cytometry.
 9. The postpartum-derived cell of claim 8wherein the cell does not spontaneously differentiate along acardiogenic, angiogenic, hemangiogenic, or vasculogenic pathway whencultured in Growth Medium.
 10. A population of cells comprising thepostpartum-derived cell of claim
 8. 11. The population of claim 10comprising from about 1% postpartum-derived cells to about 10%postpartum cells.
 12. The population of claim 11 comprising about 50%postpartum-derived cells.
 13. The population of claim 12 comprising atleast 90% postpartum-derived cells.
 14. The population of claim 13comprising substantially only postpartum-derived cells.
 15. Thepopulation of claim 14 comprising a clonal cell line ofpostpartum-derived cells.
 16. A cell lysate prepared from the populationof claim
 10. 17. The population of claim 10 incubated in the presence ofone or more factors which stimulate stem cell differentiation along acardiogenic, angiogenic, hemangiogenic, or vasculogenic pathway.
 18. Thepopulation of claim 17 wherein the factors comprise at least one of ademethylation agent, a member of BMP, FGF, TAK, GATA, Csx, NK, MEF2,ET-1, and Wnt factor families, Hedgehog, Csx/Nkx-2.5, and anti-Wntfactors.
 19. The population of claim 18 wherein the demethylation agentcomprises an inhibitor of DNA methyltransferase or an inhibitors of ahistone deacetylase, or inhibitors of a repressor complex.
 20. Thepopulation of claim 18 wherein the demethylation agents comprise atleast one of 5-azacytidine, 5-aza-2′-deoxycytidine, DMSO, chelerythrinechloride, retinoic acid or salts thereof, 2-amino-4-(ethylthio)butyricacid, procainamide, and procaine.
 21. The population of claim 10 that isseed onto a matrix to form a matrix-cell complex.
 22. The population ofclaim 21 wherein the matrix is a scaffold.
 23. The population of claim22 wherein the scaffold is bioabsorbable.
 24. The population of claim 23wherein the scaffold comprises at least one other cell type.
 25. Thepopulation of claim 24 wherein the other cell type is a stem cell.
 26. Atherapeutic cell composition comprising a pharmaceutically-acceptablecarrier and post-partum-derived cells derived from human postpartumtissue substantially free of blood, said cells capable of self-renewaland expansion in culture and having the potential to differentiate intocells of cardiomyocyte phenotypes; the cells capable of growth in anatmosphere containing oxygen from about 5% to at least about 20%;wherein said cells: require L-valine for growth; have the potential forat least about 40 doublings in culture; attach and expand on a coated oruncoated tissue culture vessel, wherein a coated tissue culture vesselcomprises a coating of gelatin, laminin, collagen, polyornithine,vitronectin, or fibronectin; produce tissue factor, vimentin, andalpha-smooth muscle actin; produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C; and do not produce any of CD31, CD34, CD45,CD117, CD 141, or HLA-DR,DP,DQ, as detected by flow cytometry.
 27. Amethod of treating a patient with a disease of the heart or circulatorysystem comprising administering the therapeutic cell composition ofclaim
 26. 28. The method of claim 27 wherein the patient has acardiomyopathy.
 29. The method of claim 28 wherein the cardiomyopathy isidiopathic.
 30. The method of claim 29 wherein the cardiomyopathy isischemic cardiomyopathy or nonischemic cardiomyopathy.
 31. The method ofclaim 30 wherein the cardiomyopathy is a nonischemic cardiomyopathyselected from dilated cardiomyopathy, hypertrophic cardiomyopathy, andrestrictive cardiomyopathy.
 32. The method of claim 27 wherein the cellsare induced along a cardiogenic, angiogenic, hemangiogenic, orvasculogenic pathway.
 33. The method of claim 32 comprising cellsinduced in vitro.
 34. The method of claim 33 comprising cells induced invitro.
 35. The method of claim 27 comprising cells that stimulate adultstem cells present in the heart to divide, or differentiate, or both.36. The method of claim 27 wherein the administering is by injection.37. The method of claim 36 wherein the injection is intracardiacinjection.
 38. The method of claim 27 wherein the cells are provided asa matrix-cell complex.
 39. The method of claim 38 wherein the matrix isa scaffold.
 40. The method of claim 39 wherein the scaffold isbioabsorbable. 41 The therapeutic cell composition of claim 26 furthercomprising the following characteristics: expression of at least one ofinterleukin 8; reticulon 1; chemokine (C-X-C motif) ligand 1 (melanomagrowth stimulating activity, alpha); chemokine (C-X-C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3; expression of atleast one of C-type (calcium dependent, carbohydrate-recognition domain)lectin, superfamily member 2 (activation-induced); Wilms tumor 1;aldehyde dehydrogenase 1 family, member A2; and renin; oxidized lowdensity lipoprotein (lectin-like) receptor 1; Homo sapiens, cloneIMAGE:4179671, mRNA, partial cds; protein kinase C, zeta; hypotheticalprotein DKFZp564F013; downregulated in ovarian cancer 1; Homo sapiensmRNA; and cDNA DKFZp547K1113 (from clone DKFZp547K1113); expression,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an ileac crest bone marrow cell, is reduced for at least oneof: short stature homeobox 2; heat shock 27 kDa protein 2; chemokine(C-X-C motif) ligand 12 (stromal cell-derived factor 1); elastin(supravalvular aortic stenosis, Williams-Beuren syndrome); Homo sapiensmRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox2 (growth arrest-specific homeobox); sine oculis homeobox homolog 1(Drosophila); crystallin, alpha B; disheveled associated activator ofmorphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1;tetranectin (plasminogen binding protein); src homology three (SH3) andcysteine rich domain; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; -neuroblastoma, suppression of tumorigenicity 1;insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, beta 7; transcriptional co-activatorwith PDZ-binding motif (TAZ); sine oculis homeobox homolog 2(Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;early growth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; transcriptionalco-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin;integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNAfull length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367protein; natriuretic peptide receptor C/guanylate cyclase C(atrionatriuretic peptide receptor C); hypothetical protein FLJ14054;Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222);BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF,HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1; and lack of secretion of atleast one of TGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC, and VEGF, asdetected by ELISA.
 42. The therapeutic cell composition of claim 41comprising about 50% postpartum-derived cells.
 43. The therapeutic cellcomposition of claim 42 comprising substantially only postpartum-derivedcells.
 44. The therapeutic cell composition of claim 43 comprising asubstantially homogeneous population of postpartum-derived cells. 45.The therapeutic cell composition of claim 44 comprising a clonal cellline of postpartum-derived cells.
 46. The therapeutic cell compositionof claim 44 wherein the substantially homogeneous population isumbilical-derived cells.
 47. The therapeutic cell composition of claim44 wherein the substantially homogeneous population is substantiallyfree of maternal cells.
 48. The therapeutic cell composition of claim 44wherein the substantially homogeneous population is placental-derivedcells.
 49. The therapeutic cell composition of claim 44 wherein thesubstantially homogeneous population is of neonatal origin.
 50. Thetherapeutic cell composition of claim 44 wherein the substantiallyhomogeneous population is of maternal origin.
 51. A method for treatinga patient with a cardiomyopathy comprising administering a therapeuticpostpartum-derived cell composition to a patient with a cardiomyopathy;and evaluating the patient for improvements in cardiac function.
 52. Themethod of claim 51 wherein the cells are induced along a cardiogenic,angiogenic, hemangiogenic, or vasculogenic pathway.
 53. The method ofclaim 51 wherein the cells stimulate adult stem cells present in theheart to divide or differentiate, or both.
 54. The method of claim 51wherein improvements include improvements in chest cardiac output (CO),cardiac index (CI), pulmonary artery wedge pressures (PAWP), and cardiacindex (CI), % fractional shortening (% FS), ejection fraction (EF), leftventricular ejection fraction (LVEF); left ventricular end diastolicdiameter (LVEDD), left ventricular end systolic diameter (LVESD),contractility (e.g. dP/dt), pressure-volume loops, measurements ofcardiac work, as well as an increase in atrial or ventricularfunctioning; an increase in pumping efficiency, a decrease in the rateof loss of pumping efficiency, a decrease in loss of hemodynamicfunctioning; and a decrease in complications associated withcardiomyopathy.
 55. The method of claim 51 wherein the administering isin vivo by transplanting, implanting, injecting, fusing, delivering viacatheter, or providing as a matrix-cell complex.
 56. The method of claim55 wherein the administering is by intracardiac injection.
 57. Themethod of claim 51 wherein the administering is to a patient who issyngeneic with the postpartum-derived cells.
 58. The method of claim 51wherein the administering is to a patient who is allogeneic
 59. Acoculture of human postpartum cells and another mammalian cell, whereinat least one of the cells is a cardiomyoblast, angioblast, orhemangioblast or induced to differentiate along a pathway leading to acardiomyoblast, angioblast or hemangioblast.
 60. A method of treating apatient with a disease of the heart or circulatory system comprisingadministering to the patient a therapeutic composition comprising one ormore of postpartum-derived cells, a conditioned medium generated bypostpartum-derived cells, a cell lysate derived from postpartum-derivedcells, a soluble cell fraction from postpartum-derived cells, anextracellular matrix from postpartum-derived cells.
 61. The method ofclaim 60 further comprising coadministering one or more of anantithrombogenic agent, an anti-inflammatory, an immunosuppressiveagent, an immunomodulatory agent, and an antiapoptotic agent.